pBluescript XR cDNA Library Construction installation Guide

pBluescript II XR cDNA Library
Construction Kit

INSTRUCTION MANUAL

Catalog #200455

BN #200455-12

Revision A.01

For In Vitro Use Only

200455-12

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pBluescript II XR cDNA Library Construction Kit

CONTENTS

Materials Provided.............................................................................................................................. 1

Storage Conditions.............................................................................................................................. 2

Additional Materials Required .......................................................................................................... 3

Reagents and Solutions..........................................................................................................3

Equipment ............................................................................................................................. 3

Introduction......................................................................................................................................... 4

pBluescript II SK (+) Vector Map......................................................................................... 5

cDNA Insert Preparation and Ligation............................................................................................. 6

Background for Preparation of cDNA Libraries ................................................................... 6

CDNA Synthesis.................................................................................................................... 7

Generation of cDNA Inserts.................................................................................................. 9

Transformation Guidelines.................................................................................................. 22

Transformation Protocol...................................................................................................... 23

Determining the Number of Transformants ........................................................................ 24

Verifying the Insert Percentage and Size ............................................................................ 25

Amplifying the pBluescript cDNA Library......................................................................... 26

Appendix I: RNA Purification and Quantification........................................................................ 28

Appendix II: Methylmercury Hydroxide Treatment .................................................................... 29

Appendix III: Alkaline Agarose Gels .............................................................................................. 30

Appendix IV: Ethidium Bromide Plate Assay— Quantitation of DNA....................................... 33

Troubleshooting ................................................................................................................................ 34

Preparation of Media and Reagents................................................................................................ 35

References .......................................................................................................................................... 37

Endnotes............................................................................................................................................. 37

MSDS Information............................................................................................................................ 37

pBluescript II XR cDNA Library Construction Kit

Caution DO NOT substitute the reagents in this kit with reagents from another kit. Component

substitution may result in lower-efficiency library construction.

MATERIALS PROVIDED

Materials provideda Quantity Storage

pBluescript II SK (+) XR vector (20 ng/ μl) [EcoR I–Xho I digested, treated with calf intestinal alkaline phosphatase (CIAP)]b 55 μl –20°C

KAN SR fragment (test insert) (10 ng/ μl, 1.3 kb) 3 μl –20°C

XL10-Gold ultracompetent cells

pUC18 DNA control plasmid (0.1 ng/μl in TE buffer) 10 μl –20°C

XL10-Gold β-Mercaptoethanol mix (β-ME) 50 μl –80°C

First-strand reagents

AccuScript reverse transcriptase (AccuScript RT) 15 μl –20°C

RNase Block Ribonuclease Inhibitor (40 U/μl) 200 U –20°C

First-strand methyl nucleotide mixture (10 mM dATP, dGTP, and dTTP plus 5 mM 5-methyl dCTP) 15 μl –20°C

First-strand buffer (10×) 75 μl –20°C

Linker–primer (1.4 μg/μl) 10 μl –20°C

Test poly(A)+ RNA (0.2 μg/μl) 5 μg –20°C

Diethylpyrocarbonate (DEPC)-treated water 500 μl –20°C

Second-strand reagents

Second-strand buffer (10×) 150 μl –20°C

Second-strand dNTP mixture (10 mM dATP, dGTP, and dTTP plus 26 mM dCTP) 30 μl –20°C

Escherichia coli RNase H (1.5 U/μl) 15 U –20°C

Escherichia coli DNA polymerase I (9.0 U/μl) 500 U –20°C

Sodium acetate (3 M) 250 μl –20°C

Blunting reagents

Blunting dNTP mixture (2.5 mM dATP, dGTP, dTTP, and dCTP) 115 μl –20°C

Cloned Pfu DNA polymerase (2.5 U/μl) 25 U –20°C

Ligation reagents

EcoR I adapters (0.4 μg/μl) 18 μg –20°C

Ligase buffer e (10×) 250 μl –20°C

rATPe (10 mM) 100 μl –20°C

T4 DNA ligasee (4 U/μl) 140 U –20°C

(Table continues on next page)

a

Enough reagents are included to generate five vector-ligated constructs.

b

After thawing, aliquot and store at –20°C. Do not pass through more than two freeze–thaw cycles. For short-term storage, store at 4°C for 1 month.

c

Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac Tet

d

On arrival, store the ultracompetent cells immediately at –80°C. Do not place the cells in liquid nitrogen.

e

These reagents are used more than once in the reaction.

Revision A.01 © Agilent Technologies, Inc. 2009.

c,d

(yellow tubes) 10 × 100 μl –80°C

R

Hte [F´ proAB lacIqZΔM15 Tn10 (TetR) Amy CamR].

pBluescript II XR cDNA Library Construction Kit 1

(table continued from the previous page)

Materials provided Quantity Storage

Phosphorylation reagents

T4 polynucleotide kinase (5 U/μl) 50 U –20°C

Ligase buffere (10×) 250 μl –20°C

rATPe (10 mM) 100 μl –20°C

Xho I digestion reagents

Xho I (40 U/μl) 600 U –20°C

Xho I buffer supplement 250 μl –20°C

Column reagents (shipped separately at 4°C)

Sepharose CL-2B gel filtration medium 10 ml 4°C

Column-loading dye 17.5 μl 4°C

STE buffer (10×) 10 ml 4°C

Connector tubing (1-inch i.d., 3/16-inch o.d., and 1/32-inch wall) 1 × 4 cm Rm Temp

e

These reagents are used more than once in the reaction.

STORAGE CONDITIONS

XL10-Gold Ultracompetent Cells: –80°C
XL10-Gold β-Mercaptoethanol Mix: –80°C
pUC18 DNA Control Plasmid: –20°C
pBluescript II XR Vector: –20°C
KAN SR Fragment (Test Insert): –20°C
Other Reagents: –20°C
Sepharose
Column-Loading Dye: 4°C
STE buffer: 4°C

®

CL-2B Gel Filtration Medium: 4°C

2 pBluescript II XR cDNA Library Construction Kit

ADDITIONAL MATERIALS REQUIRED

Certain reagents recommended in this instruction manual are potentially dangerous and present the
following hazards: chemical (DEPC, phenol, chloroform, methylmercury hydroxide, and sodium
hydroxide), radioactive (
systems). The researcher is advised to take proper precautions and care with these hazards and to
follow the safety recommendations from each respective manufacturer.

Reagents and Solutions

Phenol–chloroform [1:1 (v/v)] and chloroform

Note Do not use the low-pH phenol from the Stratagene RNA Isolation Kit because this phenol

is acidic and may denature the DNA.

Ethanol (EtOH) [70%, 80%, and 100% (v/v)]
Sterile distilled water (dH

32

[α-

P]Deoxynucleoside triphosphate ([α-32P]dNTP) (800 Ci/mmol) ([α-32P]dATP,

32

[α-

P]dGTP, or [α-32P]dTTP may be used; do not use [α-32P]dCTP)

SeaPrep agarose [FMC, Rockland, Maine (Catalog #50302)]

Equipment

Ribonuclease (RNase)-free microcentrifuge tubes
Disposable plastic 10-ml syringes, sterile (e.g., B-D
Disposable 18 gauge, 1½-inch needles, sterile (e.g., B-D
Disposable plastic 1-ml pipets, negatively graduated and sterile [e.g., Falcon

pipet (1 ml)]
Pasteur pipet
Portable radiation monitor (Geiger counter)
Water baths (8°, 12°, 16°, 30°, 37°, 42°, 65°, 70°, and 72°C)
Microcentrifuge
Micropipet and micropipet tips
Vacuum evaporator
Incubator (30° and 37°C)

®

Falcon

2059 polypropylene tubes

32

P-labeled radioisotope), or physical (high-voltage electrophoresis

O)

2

®

10-cc syringe with a Luer Lok® tip)

®

PrecisionGlide® needle)

®

7520 1-ml serological

pBluescript II XR cDNA Library Construction Kit 3

INTRODUCTION

The construction of cDNA libraries is fundamental in discovering new
genes and assigning gene function. cDNA libraries constructed directly into
plasmid vectors are convenient for plasmid-based functional screening,
expressed sequence tagged (EST) sequencing, normalization, and
subtraction techniques. The pBluescript II library construction kit is
designed for generating directional cDNA libraries in the pBluescript II SK
(+) vector. Libraries constructed in the pBluescript II SK (+) vector can be
screened with a DNA probe or an antibody probe in E. coli. When screening
libraries with a functional assay, directional cloning increases the
probability of finding clones two-fold when compared to non-directional
libraries. With this system, cDNA is synthesized and fractionated by a drip­column method to increase clonal yield and cDNA insert length. cDNA
inserts are ligated into the pBluescript II SK (+) vector to generate a plasmid
library. The cDNA plasmid library is then transformed into XL10-Gold
ultracompetent cells. Libraries generated with XL10-Gold cells increase the
probability of obtaining full-length clones.

The pBluescript II SK (+) vector is a high-copy-number pUC-based plasmid
with ampicillin resistance and the convenience of blue–white color
selection. The vector supplied in this kit is predigested with EcoR I and Xho
I restriction enzymes. The multiple cloning site (MCS) contains unique
restriction enzyme recognition sites organized with alternating 5´ and 3´
overhangs to allow serial exonuclease III/mung bean nuclease deletions.

1

T3
and T7 RNA polymerase promoters flank the polylinker for in vitro RNA
synthesis. The choice of promoter used to initiate transcription determines
which strand of the DNA insert will be transcribed. BssH II sites flank the
T3 and T7 promoters. This rare six-base restriction enzyme allows the insert
plus the RNA promoters to be excised and used for gene mapping.

Expression in the pBluescript II SK (+) vector is driven by the lac promoter,
which is repressed in the presence of the LacI protein and is inducible by

isopropyl-β-

D-thio-galactopyranoside (IPTG). In bacteria expressing the

lacZΔM15 mutation and lacI, such as XL10-Gold cells, colonies containing
vector without insert will be blue in the presence of 5-bromo-4-chloro-3-

indoyl-β-

D-galactopyranoside (X-gal) and IPTG. Ampicillin-resistant

colonies containing vector with insert will be white and can express the
inserted gene as a fusion protein. The MCS and T7 and T3 RNA polymerase
promoter sequences are present in the N-terminal portion of a lacZ gene
fragment. There are 36 amino acids from the MET sequence to the EcoR I
site. There are a total of 131 amino acids, but this is interrupted by the large
MCS.

The pBluescript II XR vector can be rescued as single-stranded (ss) DNA.
pBluescript II phagemids contain a 454-bp filamentous f1 phage intergenic
region (M13 related), which includes the 307-bp origin of replication. The
(+) orientation of the f1 intergenic region allows the rescue of lacZ’ sense
ssDNA by a helper phage. This ssDNA can be used for dideoxynucleotide
sequencing (Sanger method) or site-directed mutagenesis. Additional
sequence and restriction site information for the pBluescript II SK (+) vector
is available from the Stratagene website (http://www.stratagene.com).

4 pBluescript II XR cDNA Library Construction Kit

pBluescript II SK (+) Vector Map

ampicillin

pBluescript II SK (+)

3.0 kb

pUC ori

pBluescript II SK (+) Multiple Cloning Site Region
(sequence shown 598–826)

BssH II

TTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGAC...

M13 –20 primer binding site

...GGTATCGATAAGCTTGATATCGAATTCCTGCAGCCCGGGGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTC...

Bsp106 I
Cla I BamH I

EcoR IEcoR V

T7 Promoter

T7 primer binding site

Spe ISma I Xba IPst IHind III

SK primer binding site...KS primer binding site

f1 (+) ori

P lac

Apa I

Kpn I

EcoO109 I
Dra II

Not I

Eag I

lacZ'

Kpn I

MCS

Sac I

Xho I

KS primer binding site...

Sac II

BstX I

Hinc II
Acc I
Sal I

Sac I

T3 Promoter

...CAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCC

T3 primer binding site

BssH II

βα

-gal -fragment

M13 Reverse primer binding site

Feature Nucleotide Position

f1 (+) origin of ss-DNA replication 135–441
β-galactosidase α-fragment coding sequence (lacZ’) 460–816

multiple cloning site 653–760

T7 promoter transcription initiation site 643

T3 promoter transcription initiation site 774

lac promoter 817–938

pUC origin of replication 1158–1825

ampicillin resistance (bla) ORF 1976–2833

FIGURE 1 The pBluescript II SK (+) phagemid vector. The complete sequence and list of restriction sites are available at

www.stratagene.com.

supplied in this kit is predigested with EcoR I and Xho I restriction enzymes.

The vector sequence is also available from the GenBank

®

database (accession #X52328). The vector

pBluescript II XR cDNA Library Construction Kit 5

CDNA INSERT PREPARATION AND LIGATION

Background for Preparation of cDNA Libraries

Complementary DNA libraries represent the information encoded in the
mRNA of a particular tissue or organism. RNA molecules are exceptionally
labile and difficult to amplify in their natural form. For this reason, the
information encoded by the RNA is converted into a stable DNA duplex
(cDNA) and then is inserted into a self-replicating plasmid vector. Once the
information is available in the form of a cDNA library, individual processed
segments of the original genetic information can be isolated and examined
with relative ease.

The pBluescript II XR cDNA library construction kit uses a hybrid
oligo(dT) linker–primer that contains an Xho I restriction site. Messenger
RNA is primed in the first-strand synthesis with the linker–primer and is
reverse-transcribed using AccuScript reverse transcriptase and 5-methyl
dCTP.

AccuScript reverse transcriptase (AccuScript RT) is a novel Moloney
murine leukemia virus reverse transcriptase (MMLV-RT) derivative
combined with a proofreading 3’-5’ exonuclease. AccuScript reverse
transcriptase delivers the highest reverse-transcription accuracy while
promoting full length cDNA synthesis. AccuScript reverse transcriptase
delivers greater than three-fold higher accuracy compared to leading reverse
transcriptases, representing a significant advancement in cDNA synthesis
accuracy. These advantages make AccuScript RT the enzyme of choice for
applications involving the preparation of accurate, full-length, cDNA
transcripts, including first-strand cDNA synthesis and library construction.

The use of 5-methyl dCTP during first-strand synthesis hemimethylates the
cDNA, which protects the cDNA from digestion with certain restriction
endonucleases such as Xho I. Therefore, on Xho I digestion of the cDNA,
only the unmethylated site within the linker–primer is cleaved.

Hemimethylated DNA introduced into an McrA
subject to digestion by the mcrA and mcrB restriction systems. Therefore, it
is necessary to initially infect an McrA
cells supplied with the system) when using the pBluescript cDNA library
construction kit. After passing the library through XL10-Gold cells, the
DNA is no longer hemimethylated and can be grown on McrA
strains (e.g., XL1-Blue strain).

+

McrB+ strain would be

–

McrB– strain (e.g., the XL10-Gold

+

McrB+

6 pBluescript II XR cDNA Library Construction Kit

CDNA Synthesis

The yield, length, and accuracy of cDNA transcripts is enhanced with the
use of AccuScript RT, an engineered version of the Moloney murine
leukemia virus reverse transcriptase combined with a proofreading 3’-5’
exonuclease. First-strand cDNA synthesis begins when AccuScript RT, in
the presence of nucleotides and buffer, finds a template and a primer. The
template is mRNA and the primer is a 50-base oligonucleotide with the
following sequence:

5´-GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAGTTTTTTTTTTTTTTTTTT-3´

"GAGA" Sequence Xho I Poly(dT)

This oligonucleotide was designed with a "GAGA" sequence to protect the
Xho I restriction enzyme recognition site and an 18-base poly(dT) sequence.
The restriction site allows the finished cDNA to be inserted into the
pBluescript vector in a sense orientation (EcoR I–Xho I). The poly(dT)
region binds to the 3´ poly(A) region of the mRNA template, and
AccuScript RT begins to synthesize the first-strand cDNA.

The nucleotide mixture for the first strand contains normal dATP, dGTP,
and dTTP plus the analog 5-methyl dCTP. The complete first strand will
have a methyl group on each cytosine base, which will protect the cDNA
from restriction enzymes used in subsequent cloning steps.

During second-strand synthesis, RNase H nicks the RNA bound to the first­strand cDNA to produce a multitude of fragments, which serve as primers
for DNA polymerase I. DNA polymerase I "nick-translates" these RNA
fragments into second-strand cDNA. The second-strand nucleotide mixture
has been supplemented with dCTP to reduce the probability of 5-methyl
dCTP becoming incorporated in the second strand. This ensures that the
restriction sites in the linker–primer will be susceptible to restriction enzyme
digestion. The uneven termini of the double-stranded cDNA are chewed
back or filled in with cloned Pfu DNA polymerase, and EcoR I adapters are
ligated to the blunt ends of the cDNA fragments. The adapters have the
sequence shown below.

5´-OH-AATTCGGCACGAGG-3´

3´-GCCGTGCTCC-P-5´

These adapters are composed of 10- and 14-mer oligonucleotides, which are
complementary to each other with an EcoR I cohesive end. The 10-mer
oligonucleotide is phosphorylated, which allows it to ligate to other blunt
termini available in the form of cDNA and other adapters. The 14-mer
oligonucleotide is kept dephosphorylated to prevent it from ligating to other
cohesive ends. After adapter ligation is complete and the ligase has been
heat inactivated, the 14-mer oligonucleotide is phosphorylated to enable its
ligation to the dephosphorylated vector.

The Xho I digestion releases the EcoR I adapter and residual linker–primer
from the 3´ end of the cDNA. These two fragments are separated on a drip

®

column containing Sepharose

CL-2B gel filtration medium. The size-

fractionated cDNA is then precipitated and ligated to the pBluescript vector.

pBluescript II XR cDNA Library Construction Kit 7

The ligated DNA is then transformed into XL10-Gold cells. Since most

E. coli strains digest DNA containing 5´-methyl dCTP, it is important to
transform into this McrA

–

McrB– strain to obtain the highest yield.

Note An outline of the pBluescript II XR cDNA library construction kit

is provided (see Figure 2). If you plan to be away from the project
for 1 or 2 days, it is best to schedule the synthesis such that the
cDNA remains in the ligation reaction. Even though the majority
of ligation is complete in the time recommended by the procedure,
the provided ligase is extremely active and will continue to find
and ligate available ends. Although most investigators wish to
produce their cDNA libraries as rapidly as possible, it is
important to remember that extended ligations and overnight
precipitations can increase the yield.

Messenger RNA template

First-strand synthesis

Second-strand synthesis

Adapter addition

mRNA

RNA

Reverse transcriptase
5-methyl dCTP
dATP, dGTP, dTTP

RNase H
DNA polymerase I
dNTPs

Eco

R I adapters

T4 DNA ligase

Xho I restriction enzyme

Xho

I digestion

Completed unidirectional cDNA

FIGURE 2 cDNA synthesis flow chart.

8 pBluescript II XR cDNA Library Construction Kit

Generation of cDNA Inserts

Notes DO NOT substitute the reagents in this kit with reagents from

another kit. Component substitution may result in lower-efficiency
library construction.

The following protocol uses radiolabeled nucleotides in control

first- and second-strand synthesis reactions to assess the quality
and the size of the cDNA synthesis products. An alternative
protocol, using SYBR Green II staining instead of
available in a Technical Note on the Stratagene website:

http://www.stratagene.com/lit_items/cDNA_Synthesis_Kit­Using_SYBR®.pdf.

32

P labeling, is

The following protocol is optimized for 5

μ

g of poly(A)+ RNA.

Protocol Guidelines

♦ The quality and quantity of the mRNA used is of fundamental

importance to the construction of a large, representative cDNA library
(see Appendix II: RNA Purification and Quantitation). The Stratagene
RNA Isolation Kit (Stratagene Catalog #200345) uses the guanidinium
thiocyanate–phenol–chloroform extraction method,
produces large amounts of undegraded RNA. To isolate mRNA, we
offer the Absolutely mRNA Purification Kit (Stratagene Catalog
#400806).

♦ If poor first-strand synthesis suggests problems associated with

secondary structure (e.g., hairpinning), treatment with methylmercury
hydroxide (CH

HgOH) is recommended to relax secondary structure

3

(see Appendix III: Methylmercury Hydroxide Treatment).

♦ It is imperative to protect the RNA from any contaminating RNases

until the first-strand cDNA synthesis is complete. Wear fresh gloves,
use newly autoclaved pipet tips, and avoid using pipet tips or
microcentrifuge tubes that have been handled without gloves.
Ribonuclease A cannot be destroyed by normal autoclaving alone.
Baking or DEPC treatment is recommended.

2

which quickly

♦ When removing aliquots of any of the enzymes used in the pBluescript

II XR cDNA synthesis protocol, flick the bottom of the tube to
thoroughly mix the enzyme solution. Do not vortex the enzyme stock
tubes.

pBluescript II XR cDNA Library Construction Kit 9

Synthesizing First-Strand cDNA

1. Preheat two water baths to 42° and 72°C, respectively.

2. Thaw the radioactive [α-

32

P]dNTP (do not use [32P]dCTP) and all
nonenzymatic first-strand components. Keep the radioactive dNTP on
ice for use in step 6 and again in the second-strand synthesis. Briefly
vortex and spin down the contents of the nonenzymatic tubes. Place the
tubes on ice.

Note AccuScript RT is temperature sensitive and should remain at

–20°C until the last moment.

3. The final volume of the first-strand synthesis reaction is 50 μl. The
volume of added reagents and enzymes is 14 μl, thus the mRNA

template and DEPC-treated water should be added in a combined
volume of 36 μl. For the control reaction, prepare the following
annealing reaction with 25 μl (5 μg) of test RNA and 11 μl of DEPC-

treated water.

4. In an RNase-free microcentrifuge tube, add the following reagents in
order:

5 μl of 10× first-strand buffer
3 μl of first-strand methyl nucleotide mixture
2 μl of linker–primer (1.4 μg/μl)
X μl of DEPC-treated water
1 μl of RNase Block Ribonuclease Inhibitor (40 U/μl)

5. Mix the reaction and then add X μl of poly(A)

+

RNA (5 μg). Mix

gently.

6. Allow the primer to anneal to the template for 10 minutes at room
temperature. During the incubation, aliquot 0.5 μl of the [α-

32

P]dNTP

(800 Ci/mmol) into a separate tube for the control.

7. Add 3 μl of AccuScript RT to the first-strand synthesis reaction. The
final volume of the first-strand synthesis reaction should now be 50 μl.

8. Mix the sample gently and spin down the contents in a microcentrifuge.

9. Transfer 5 μl of the first-strand synthesis reaction to the separate

tube containing the 0.5 μl of the [α-

32

P]dNTP (800 Ci/mmol). This

radioactive sample is the first-strand synthesis control reaction.

10. Incubate the first-strand synthesis reactions, including the control
reaction, at 42°C for 1 hour.

10 pBluescript II XR cDNA Library Construction Kit

11. Prepare a 16°C water bath for second-strand synthesis. If a water bath
with a cooling unit is not available, use a large Styrofoam

®

container
with a lid. Fill the container three-quarters full with water and adjust
the temperature to 16°C with ice. Cover the container with a lid.

12. After 1 hour, remove the first-strand synthesis reactions from the 42°C
water bath. Place the nonradioactive first-strand synthesis reaction on
ice. Store the radioactive first-strand synthesis control reaction at
–20°C until ready to resolve by electrophoresis on an alkaline agarose
gel (see Appendix III: Alkaline Agarose Gels). Both first- and second­strand reactions should be run on the same gel after the second-strand
reaction has been blunted and resuspended in the EcoR I adapters (see
step 17 in Blunting the cDNA Termini).

Synthesizing Second-Strand cDNA

1. Thaw all nonenzymatic second-strand components. Briefly vortex and
spin in a micro-centrifuge before placing the tubes on ice.

Note It is important that all reagents be <16°C when the DNA

polymerase I is added.

2. Add the following components in order to the 45-μl nonradioactive,
first-strand synthesis reaction on ice:

20 μl of 10× second-strand buffer
6 μl of second-strand dNTP mixture
114 μl of sterile dH
2 μl of [α-

32

O (DEPC-treated water is not required)

2

P]dNTP (800 Ci/mmol)

3. Add the following enzymes to the second-strand synthesis reaction:

2 μl of RNase H (1.5 U/μl)
11 μl of DNA polymerase I (9.0 U/μl)

4. Gently mix the contents of the tube, spin the reaction in a
microcentrifuge, and incubate for 2.5 hours in a 16°C water bath.
Check the water bath occasionally to ensure that the temperature does
not rise above 16°C. Temperatures above 16°C can cause the formation
of hairpin structures, which are unclonable and interfere with the
efficient insertion of correctly synthesized cDNA into the prepared
vector.

5. After second-strand synthesis for 2.5 hours at 16°C, immediately place
the tube on ice.

pBluescript II XR cDNA Library Construction Kit 11

Blunting the cDNA Termini

1. Add the following to the second-strand synthesis reaction:

23 μl of blunting dNTP mix
2 μl of cloned Pfu DNA polymerase

2. Gently mix the reaction and spin down in a microcentrifuge. Incubate
the reaction at 72°C for 30 minutes. Do not exceed 30 minutes!!

3. Thaw the 3 M sodium acetate.

Note Since radioactivity can leak out between the lid and body of

some micro-centrifuge tubes during the vortexing and
precipitation steps, wrap a small piece of Parafilm

®

laboratory film around the rim of the microcentrifuge tube to
prevent leakage.

4. Remove the reaction and add 200 μl of phenol–chloroform [1:1 (v/v)]
and vortex.

Note Do not use the low-pH phenol from the Stratagene RNA

Isolation Kit because this phenol is acidic and may denature
the DNA. The phenol must be equilibrated to pH 7–8.

5. Spin the reaction in a microcentrifuge at maximum speed for 2 minutes
at room temperature and transfer the upper aqueous layer, containing
the cDNA, to a new tube. Be careful to avoid removing any interface
that may be present.

6. Add an equal volume of chloroform and vortex.

7. Spin the reaction in a microcentrifuge at maximum speed for 2 minutes
at room temperature and transfer the upper aqueous layer, containing
the cDNA, to a new tube.

8. Precipitate the cDNA by adding the following to the saved aqueous
layer:

20 μl of 3 M sodium acetate
400 μl of 100% (v/v) ethanol

Vortex the reaction.

9. Precipitate overnight at –20°C.

10. In order to orient the direction of precipitate accumulation, place a
mark on the microcentrifuge tube or point the tube hinge away from the
center of the microcentrifuge as an indicator of where the pellet will
form.

12 pBluescript II XR cDNA Library Construction Kit

11. Spin in a microcentrifuge at maximum speed for 60 minutes at 4°C.

12. Avoid disturbing the pellet and carefully remove and discard the
radioactive supernatant in a radioactive waste container.

Note The conditions of synthesis and precipitation produce a large

white pellet. The pellet accumulates near the bottom of the
microcentrifuge tube and may taper up along the marked side
of the tube.

13. Gently wash the pellet by adding 500 μl of 70% (v/v) ethanol to the
side of the tube away from the precipitate. Do not mix or vortex!

14. Spin in a microcentrifuge at maximum speed for 2 minutes at room
temperature with the orientation marked as in step 10.

15. Aspirate the ethanol wash and dry the pellet by vacuum centrifugation.

16. Resuspend the pellet by gentle pipetting in 9 μl of EcoR I adapters and
incubate at 4°C for at least 30 minutes to allow the cDNA to resuspend.
To ensure that the cDNA is completely in solution, transfer the cDNA
to a fresh microcentrifuge tube. Monitor the now empty tube with a
handheld Geiger counter. If the cDNA is in solution, fewer counts
should remain in the empty tube.

17. Transfer 1 μl of this second-strand synthesis reaction to a separate

tube. This radioactive sample is the second-strand synthesis control
reaction. We strongly recommend running the samples of the first- and

second-strand synthesis reactions on an alkaline agarose gel at this
point. It is important to determine the size range of the cDNA and the
presence of any secondary structure (see Appendix IV: Alkaline
Agarose Gels).

Note The second-strand synthesis reaction can be stored overnight

at –20°C.

Ligating the EcoR I Adapters

1. Add the following components to the tube containing the blunted
cDNA and the EcoR I adapters:

1 μl of 10× ligase buffer
1 μl of 10 mM rATP
1 μl of T4 DNA ligase (4 U/μl)

2. Spin down the volume in a microcentrifuge and incubate overnight at
8°C. Alternatively, the ligations can be incubated at 4°C for 2 days.

3. In the morning, heat inactivate the ligase by placing the tubes in a 70°C
water bath for 30 minutes.

pBluescript II XR cDNA Library Construction Kit 13

Phosphorylating the EcoR I Ends

1. After the ligase is heat inactivated, spin the reaction in a
microcentrifuge for 2 seconds. Cool the reaction at room temperature
for 5 minutes.

2. Phosphorylate the adapter ends by adding the following components:

1 μl of 10× ligase buffer
2 μl of 10 mM rATP
5 μl of sterile water
2 μl of T4 polynucleotide kinase (5.0 U/μl)

3. Incubate the reaction for 30 minutes at 37°C.

4. Heat inactivate the kinase for 30 minutes at 70°C.

5. Spin down the condensation in a microcentrifuge for 2 seconds and
allow the reaction to equilibrate to room temperature for 5 minutes.

Digesting with Xho I

1. Add the following components to the reaction:

28 μl of Xho I buffer supplement
3 μl of Xho I (40 U/μl)

2. Incubate the reaction for 1.5 hours at 37°C.

3. Add 5 μl of 10× STE buffer and 125 μl of 100% (v/v) ethanol to the
microcentrifuge tube.

4. Precipitate the reaction overnight at –20°C.

5. Following precipitation, spin the reaction in a microcentrifuge at
maximum speed for 60 minutes at 4°C.

6. Discard the supernatant, dry the pellet completely, and resuspend the
pellet in 14 μl of 1× STE buffer.

7. Add 3.5 μl of the column-loading dye to each sample.

The sample is now ready to be run through a drip column containing
Sepharose CL-2B gel filtration medium (see Size Fractionating).

14 pBluescript II XR cDNA Library Construction Kit

Size Fractionating

Before attempting the experimental protocols outlined within this section,
please read this section in its entirety in order to become familiar with the
procedures. Review of the Troubleshooting section may also prove helpful.
The drip columns should be prepared and the cDNA should be eluted in
1 day. Because a full day is required to complete these procedures, gathering
all necessary materials in advance is recommended (see the Equipment
section in Additional Materials Required).

Assembling the Drip Column

1. Perform the following preparatory steps while assembling the drip
columns:

a. Remove the Sepharose CL-2B gel filtration medium and the

10× STE buffer from refrigeration and equilibrate the two
components to room temperature.

b. Prepare 50 ml of 1× STE buffer by diluting 10× STE buffer 1:10

in sterile water.

2. Assemble the drip columns as outlined in the following steps (see
Figure 3 for a diagram of the final setup):

Note Wear gloves while assembling the drip columns.

a. Remove the plastic wrapper from the top of a sterile 1-ml pipet.

b. Using a sterile needle or a pair of fine-tipped forceps, carefully

tease the cotton plug out of each pipet, leaving a piece of the
cotton plug measuring ~3–4 mm inside. Cut off the external
portion of the cotton plug.

c. Push the remaining 3- to 4-mm piece of the cotton plug into the

top of each pipet with the tip of the needle or forceps.

d. Cut a small piece of the connecting tubing measuring ~8 mm. Use

this small tube to connect the 1-ml pipet to the 10-ml syringe. First
attach one end of the connecting tube to the pipet and then connect
the other end to the syringe. There should be no gap between the
pipet and the syringe when joined by the connecting tube.

Note The inside diameter of the connecting tubing (~1/8-inch

i.d.) snugly connects most disposable 1-ml pipets and the

®

ends of all B-D

10-cc syringes with the Luer Lok® tips.

pBluescript II XR cDNA Library Construction Kit 15

Syringe (10-ml)

Pipet (1-ml)

10

9
8

7
6
5
4

3
2
1

c

c

Connecting tube (~8-mm)

1in 1/10ml TD 20°

-.4

-.3

-.2

-.1

.7

–.4 ml graduation

.8

.9

Cotton plug

FIGURE 3 Assembly of the drip columns.

e. Rapidly and forcefully push the plunger into the syringe to thrust

the cotton plug down into the tip of the pipet.

Note It may take several attempts to drive the cotton all the

way down into the tip of the pipet. However, pushing the
cotton plug as far down into the pipet tip as possible is
important in order to achieve optimal separation of the
cDNA fractions.

f. Remove the plunger from the syringe. Because the syringe

functions as a buffer reservoir for the drip column, leave the
syringe firmly attached to the pipet throughout the remainder of
the size fractionation procedure.

3. Locate a support for the assembled drip column. Butterfly clamps or a
three-fingered clamp on a ring stand can be used.

16 pBluescript II XR cDNA Library Construction Kit

Loading the Drip Column

1. Load the drip column with a uniform suspension of Sepharose CL-2B
gel filtration medium as outlined in the following steps:

a. Immediately prior to loading the drip column, gently mix the

Sepharose CL-2B gel filtration medium by inversion until the
resin is uniformly suspended.

b. Place the column in the ring stand. Fill a glass Pasteur pipet with

~2 ml of 1× STE buffer. Insert the pipet as far into the drip column
as possible and fill the column with the buffer.

Note If the 1× STE buffer flows too quickly through the

column, stem the flow by affixing a yellow pipet tip to the
end of the column. Make sure to remove the pipet tip
prior to loading the column with the Sepharose CL-2B
gel filtration medium.

If bubbles or pockets of air become trapped in the STE
buffer while filling the column, remove the trapped air
prior to packing the column with the resin. To remove
the bubbles or air, re-insert the Pasteur pipet into the
top of the column and gently pipet the STE buffer in and
out of the pipet until the trapped air escapes through the
top of the column.

c. Immediately add a uniform suspension of Sepharose CL-2B gel

filtration medium to the column with a Pasteur pipet by inserting
the pipet as far into the column as possible. As the resin settles,
continue adding the Sepharose CL-2B gel filtration medium. Stop
adding the resin when the surface of the packed bed is ¼ inch
below the “lip of the pipet.” The lip of the pipet is defined as the
point where the pipet and the syringe are joined.

Notes If air bubbles form as the resin packs, use a Pasteur

pipet as described in step 1b to remove the blockage.
Failure to remove bubbles can impede the flow of the
column and result in a loss of the cDNA.

If the preparation of Sepharose CL-2B gel filtration

medium settles and becomes too viscous to transfer from
the stock tube to the column, add a small volume
(~1–5 ml) of 1× STE buffer to resuspend the resin.

pBluescript II XR cDNA Library Construction Kit 17

2. Wash the drip column by filling the buffer reservoir (i.e., the syringe)
with a minimum of 10 ml of 1× STE buffer. As the column washes, the
buffer should flow through the drip column at a steady rate; however, it
may take at least 2 hours to complete the entire wash step. After
washing, do not allow the drip column to dry out, because the resin
could be damaged and cause sample loss. If this occurs, pour another
column.

Note If a free flow of buffer is not observed, then bubbles or

pockets of air have become trapped in the drip column. In
this case, the column must be repacked. If cDNA is loaded
onto a column on which a free flow of buffer is not observed,
the sample could become irretrievably lost.

3. When ~50 μl of the STE buffer remains above the surface of the resin,
immediately load the cDNA sample using a pipettor. Gently release the
sample onto the surface of the column bed, but avoid disturbing the
resin as this may affect cDNA separation.

4. Once the sample enters the Sepharose CL-2B gel filtration medium, fill
the connecting tube with buffer using a pipettor.

Note Do not disturb the bed while filling the connecting tube with

buffer.

Gently add 3 ml of 1× STE buffer to the buffer reservoir by trickling

the buffer down the inside wall of the syringe. Do not squirt the buffer
into the reservoir because this will disturb the resin, resulting in loss of
the sample.

5. As the cDNA sample elutes through the column, the dye will gradually
diffuse as it migrates through the resin. Because the dye is used to
gauge when the sample elutes from the column, monitor the progress of
the dye, or the cDNA sample could be irretrievably lost.

18 pBluescript II XR cDNA Library Construction Kit

Collecting the Sample Fractions

The drip column containing the Sepharose CL-2B gel filtration medium
separates molecules on the basis of size. Large cDNA molecules elute first
followed by smaller cDNA and finally unincorporated nucleotides. Using a
handheld monitor, two peaks of radioactivity can generally be detected
during the course of elution. The first peak to elute from the column
represents the cDNA. Due to the conditions of label incorporation during
second-strand synthesis, the cDNA is not extremely radioactive; therefore,
the counts per second may be barely above background levels. In contrast,
the second peak to elute from the column is highly radioactive as this is the
unincorporated radioactive nucleotides. Although this material elutes from
the column in parallel with the dye, unincorporated nucleotides are usually
not collected because the cDNA has already eluted from the column.

For standard cDNA size fractionation (>400 bp), collect ~12 fractions using
the procedure described in this section. The progression of the leading edge
of the dye through the column will be used as a guideline to monitor
collection; however, the drops collected from the column should be
monitored for radioactivity using a handheld Geiger counter. Until the
fractions have been assessed for the presence of cDNA on a 5%
nondenaturing acrylamide gel (see Preparation of Media and Reagents), do
not discard any fractions based on the quantity of radioactivity detected.

1. Using a fresh microcentrifuge tube to collect each fraction, begin
collecting three drops per fraction when the leading edge of the dye
reaches the –0.4-ml gradation on the pipet.

2. Continue to collect fractions until the trailing edge of the dye reaches
the 0.3-ml gradation. A minimum of 12 fractions, each containing
~100 μl (i.e., three drops), should be collected. Alternatively, fractions
can be collected until the radioactive-free nucleotides begin to elute. In
either case, monitor the fractions for the presence of radioactivity to
determine whether the cDNA has eluted successfully. If no counts are
detected, continue collecting the fractions until the peak of
unincorporated nucleotides is recovered.

3. Before processing the fractions and recovering the size-fractionated
cDNA, remove 8 μl of each collected fraction and save for later

analysis. These aliquots should be electrophoresed on a 5%
nondenaturing acrylamide gel and exposed to x-ray film overnight

to assess the effectiveness of the size fractionation and to determine
which fractions will be used for ligation.

pBluescript II XR cDNA Library Construction Kit 19

Processing the cDNA Fractions

In this section of the size fractionation procedure, the fractions collected
from the drip column are extracted with phenol–chloroform and are
precipitated with ethanol to recover the size-selected cDNA. The purpose of
the organic extractions is to remove contaminating proteins; of particular
concern is kinase, which can be carried over from previous steps in the
synthesis. Because kinase often retains activity following heat treatment, it
is necessary to follow the extraction procedures.

1. Begin extracting the remainder of the collected fractions by adding an
equal volume of phenol–chloroform [1:1 (v/v)].

2. Vortex and spin in a microcentrifuge at maximum speed for 2 minutes
at room temperature. Transfer the upper aqueous layer to a fresh
microcentrifuge tube.

3. Add an equal volume of chloroform.

4. Vortex and spin in a microcentrifuge at maximum speed for 2 minutes
at room temperature. Transfer the upper aqueous layer to a fresh
microcentrifuge tube.

5. To each extracted sample, add a volume of 100% (v/v) ethanol that is
equal to twice the individual sample volume.

Note The 1× STE buffer contains sufficient NaCl for precipitation.

6. Precipitate overnight at –20°C.

7. Spin the sample in the microcentrifuge at maximum speed for
60 minutes at 4°C. Transfer the supernatant to another tube. To ensure
that the cDNA has been recovered, use a handheld Geiger counter to
check the level of radioactivity present in the pellet. If the majority of
the radiation is detected in the supernatant, repeat the centrifugation
step; otherwise, discard the supernatant.

8. Carefully wash the pellet with 200 μl of 80% (v/v) ethanol, ensuring
that the pellet remains undisturbed. Do not mix or vortex! Spin the
sample in a microcentrifuge at maximum speed for 2 minutes at room
temperature. Remove the ethanol and verify that the pellet has been
recovered by visual inspection or with the handheld Geiger counter.
Vacuum evaporate the pellet for ~5 minutes or until dry. Do not dry the
pellet beyond the point of initial dryness or the cDNA may be difficult
to solubilize.

9. Using a handheld Geiger counter verify that the cDNA has been
recovered and record the number of counts per second (cps) that is
detected for each fraction.

10. If <30 cps is detected, resuspend each cDNA pellet in 3.5 μl of sterile
water. If the value is >30 cps, resuspend the cDNA in 5.5 μl of sterile

water. Mix by pipetting up and down.

20 pBluescript II XR cDNA Library Construction Kit

Quantitate the cDNA before proceeding (see Appendix IV: Ethidium
Bromide Plate Assay—Quantitation of DNA). Best results are usually

obtained by ligating 10 ng of cDNA/20 ng of vector. Place the remaining
cDNA at –20°C for short term storage only. The cDNA is most stable after
ligation into the vector and may be damaged during long-term storage.

Ligating cDNA into the Plasmid Vector

We recommend performing a pilot ligation and transformation for each
sample to establish the ligation efficiency of the test insert and cDNA with
the pBluescript vector. The ligation and transformation reactions may then
be scaled up and optimized to achieve a primary library consisting of a
target number of transformants.

1. Set up a control ligation to ligate the test insert into the pBluescript
vector as follows:

1.0 μl of the pBluescript vector (20 ng)

1.0 μl of KAN SR test insert (10 ng)

0.5 μl of 10× ligase buffer

0.5 μl of 10 mM rATP (pH 7.5)

1.5 μl of water

Then add

0.5 μl of T4 DNA ligase (4 U/μl)

2. To prepare the sample ligation, add the following components:

X μl of resuspended cDNA (~10 ng)

0.5 μl of 10× ligase buffer

0.5 μl of 10 mM rATP (pH 7.5)

1.0 μl of the pBluescript vector (20 ng/μl)
X μl of water for a final volume of 4.5 μl

Then add

0.5 μl of T4 DNA ligase (4 U/μl)

Note In all ligations, the final glycerol content should be less than

5% (v/v). Do not exceed 5% (v/v) glycerol!

3. Incubate the reaction tubes overnight at 12°C or for up to 2 days at 4°C.

4. After ligation is complete, transform XL10-Gold ultracompetent cells
with the ligation reactions. XL10-Gold is a McrA

–

and McrB– strain

and does not restrict methylated DNA. Use of any other cell line may
result in dramatically reduced transformation efficiency.

pBluescript II XR cDNA Library Construction Kit 21

Transformation Guidelines

Storage Conditions

Ultracompetent cells are sensitive to even small variations in temperature
and must be stored at the bottom of a –80°C freezer. Transferring tubes from
one freezer to another may result in a loss of efficiency. Ultracompetent
cells should be placed at –80°C directly from the dry ice shipping container.

Aliquoting Cells

When aliquoting, keep ultracompetent cells on ice at all times. It is essential
that the Falcon
are thawed and that the cells are aliquoted directly into the prechilled tubes.

It is also important to use at least 100 μl of ultracompetent
cells/transformation. Using a smaller volume will result in lower
efficiencies.

Use of Falcon® 2059 Polypropylene Tubes

It is important that Falcon® 2059 polypropylene tubes are used for the
transformation protocol, since other tubes may be degraded by the

β-mercaptoethanol used in step 3 of the Transformation Protocol. In
addition, the incubation period during the heat-pulse step is critical and has
been optimized specifically for the thickness and shape of the Falcon 2059
polypropylene tubes.

®

2059 polypropylene tubes are placed on ice before the cells

Use of β-Mercaptoethanol

β-Mercaptoethanol (β-ME) has been shown to increase transformation

efficiency. The XL10-Gold β-Mercaptoethanol mix provided in this kit is
diluted and ready to use. For optimum efficiency, use 4 μl of the β-ME mix.
(Using an alternative source of β-ME may reduce transformation

efficiency.)

Length and Temperature of the Heat Pulse

There is a defined window of highest efficiency resulting from the heat
pulse during transformation. Optimal efficiencies are observed when cells
are heat pulsed for 30 seconds. Heat pulsing for at least 30 seconds is
recommended to allow for slight variations in the length of incubation.
Efficiencies decrease when incubating for <30 seconds or for >40 seconds.
Do not exceed 42°C.

22 pBluescript II XR cDNA Library Construction Kit

Transformation Protocol

1. Thaw the XL10-Gold ultracompetent cells on ice.

2. Gently mix the cells by hand. Aliquot 100 μl of the cells into a
prechilled 15-ml Falcon 2059 polypropylene tube.

3. Add 4 μl of the XL10-Gold β-ME mix provided with the kit to the
100 μl of bacteria. (Using an alternative source of β-ME may reduce

transformation efficiency.)

4. Swirl the contents of the tube gently. Incubate the cells on ice for
10 minutes, swirling gently every 2 minutes.

5. Add the entire ligation reaction (from step 3 of Ligating cDNA into the
Plasmid Vector) to the cells and swirl gently.

Note As a transformation control, add 1

(diluted 1:10 in high quality water) to another 100-

μ

l of pUC18 plasmid

μ

l aliquot

of the cells and swirl gently.

6. Incubate the tubes on ice for 30 minutes.

7. Preheat NZY

+

broth§ in a 42°C water bath for use in step 10.

8. Heat pulse the tubes in a 42°C water bath for 30 seconds. The duration
and temperature of the heat pulse is critical for obtaining the highest
efficiencies. Do not exceed 42°C.

9. Incubate the tubes on ice for 2 minutes.

10. Add 0.9 ml of preheated (42°C) NZY

+

broth to each tube and incubate

the tubes at 37°C for 1 hour with shaking at 225–250 rpm.

Notes For plating quantities of pilot ligations see Determining the

Number of Transformants.

The cells may be concentrated by centrifuging at 1000 rpm for
10 minutes if desired. Resuspend the pellet in 200

μ

l of NZY+

broth and plate.

When transforming the control pUC18 DNA, plate 5
transformation mixture in a 200-

§

LB–ampicillin agar plates.

μ

l pool of NZY+ broth on

250 colonies may be expected from each 5-

≥

transformation to yield

5×109cfu/μg.

μ

l of the

μ

l control

§

See Preparation of Media and Reagents.

pBluescript II XR cDNA Library Construction Kit 23

Determining the Number of Transformants

Plating

1. Plate 1 μl and 10 μl of each 1-ml pilot transformation onto
LB-ampicillin agar plates.

2. Plate 1 μl and 10 μl of the kanamycin-resistant test insert
transformation on LB-kanamycin agar plates.

Greater than 20 colonies should be observed from the 1-μl plating of the test
insert transformation.

Count the number of ampicillin-resistant colonies on the 1-μl plate (from
step 1 above) and multiply that number by 1000.

Example 200 colonies/

Count the number of ampicillin-resistant colonies on the 10-μl plate (from
step 1 above) and multiply that number by 100.

Example 2000 colonies/10

μ

l × 1000 μl = 2.0 × 105 total cfu.

μ

l × 1000 μl = 2.0 × 105 total cfu.

Scaling Up the Ligations and Transformations

1. Perform individual ligations according to the ligation protocol to reach
the target primary library size desired. The number of ligations
necessary may vary with each insert and should be based upon
efficiencies realized with each pilot reaction.

2. Transform 100 μl of XL10-Gold ultracompetent cells individually with
each ligation reaction (see Transformation Protocol).

3. Pool the individual transformation reactions after the one hour
incubation (step 10 of Transformation Protocol).

Note This pool is the primary library. Store the primary library at

4°C and amplify as soon as possible (see Amplifying the

pBluescript cDNA Library).

4. Plate 1 μl and 10 μl of the pooled transformations onto selection plates
to determine the total number of primary transformants.

24 pBluescript II XR cDNA Library Construction Kit

Verifying the Insert Percentage and Size

Individual colonies can be examined to determine the percentage of vectors
with inserts and the average insert size by either PCR directly from the
colony with T3 and T7 primers or by restriction analysis of individually
prepared plasmid DNA.

The XL10-Gold strain allows blue-white selection for the pBluescript II
vector due to lacZΔM15 complementation of the F´ episome. The color
selection may be seen when plating on LB plates containing 100 μg/ml of
ampicillin, 80 μg/ml of fresh X-gal, and 20 mM IPTG. Alternatively, plates
for color selection can be made by spreading 100 μl of 40 mM IPTG and
100 μl of 2% X-gal on LB-ampicillin plates 30 minutes prior to plating

transformants. X-gal should be prepared in dimethyl formamide and IPTG
prepared in sterile, distilled H
Colonies containing vectors without inserts will be blue after incubation for
12–18 hours at 37°C. Colonies with vectors containing inserts will remain
white. Further enhancement of the blue color may be obtained by placing
plates at 4°C for 2 hours following overnight growth at 37°C.

2

O (store stock solutions at –20°C until use.)

pBluescript II XR cDNA Library Construction Kit 25

Amplifying the pBluescript cDNA Library

The primary library may now be plated and screened; however,
amplification of the library is desirable to produce a large and stable
quantity of the library. Do not perform more than one round of
amplification, as slower-growing clones may be significantly
underrepresented.

We recommend amplifying plasmid libraries in 500-ml bottles of 2× LB
agarose
amplified in suspension which allows for three-dimensional, uniform colony
growth. This reduces the potential for under-representation of particular
clones due to the overgrowth of some colonies during the expansion process
that accompanies direct plating methods.

Note Each 500-ml bottle of 2× LB agarose can accommodate ~5 × 10

§

using the semi-solid amplification method.

primary cfu. To amplify a library of 1 × 10
bottles are necessary.

3,4

The libraries are

6

primary cfu, two

5

1. On a heated stir plate, using a large stir bar, combine 1.35 g of SeaPrep
agarose with 450 ml of 2× LB

§

in each 500-ml autoclavable bottle. Stir

until the agarose is in solution.

2. Autoclave the bottles and stir bars for 30 minutes.

3. Allow the bottles to cool to 37°C in a water bath (~1 hour).

4. Transfer the bottles to a stir plate and add 100 μg/ml of ampicillin.

5

5. Add up to 5 × 10

cfu/bottle of primary library and stir for several

minutes.

Note The bottles must be handled very carefully at this stage.

Avoid swirling or bumping the bottles as this may cause the
microcolonies to fall out of suspension. The bottles must be
incubated without disturbance or representation of the
amplified library may be compromised.

6. Tighten the bottle caps and incubate the bottles for 1 hour in an ice
water bath (0°C). The water level in the ice bath should be even with
the media level in the bottle.

7. Carefully remove the bottles from the ice bath and gently dry the
bottles. Loosen the bottle caps and incubate for 40–45 hours at 30°C.
(Incubation at 30°C reduces under-representation of slower growing
clones.)

8. Pour the contents of the bottles into sterile 250-ml centrifuge bottles
and spin at 10,000 × g for 20 minutes at room temperature.
(Equilibrate the rotor to room temperature several hours prior to
centrifugation. Using rotors stored at 4°C will cause the agar to
solidify.)

§

See Preparation of Media and Reagents.

26 pBluescript II XR cDNA Library Construction Kit

9. Remove the semi-solid agarose supernatant and resuspend the pellets in
25 ml of 2× LB–glycerol (12.5%)

§

per 250-ml centrifuge bottle.

Remove 100 μl for estimation of library titer and further
characterization. Pipet the remainder into 1-ml aliquots. Store at –80°C.

10. Perform 6 serial dilutions with 100 μl of the amplified library diluted
into 900 μl of LB medium. Plate 10 μl of the 10

–5

and 10–6 dilutions

onto selection plates. Amplification of a primary library containing

6

1 × 10
1 × 10

total transformants should result in a stable library of at least

9

total transformants.

§

See Preparation of Media and Reagents.

pBluescript II XR cDNA Library Construction Kit 27

APPENDIX I: RNA PURIFICATION AND QUANTIFICATION

RNA Purification

The Stratagene RNA Isolation Kit, using the guanidinium thiocyanate–
phenol–chloroform extraction method,
RNA isolation. This method is rapid, yet it produces large amounts of high­quality, undegraded RNA.

Although AccuScript RT is not inhibited by ribosomal RNA (rRNA) and
transfer RNA (tRNA) contamination, it is advisable to select the poly(A)
fraction. The amounts of rRNA and tRNA vastly outnumber the mRNA and
will decrease the efficiency of the cDNA synthesis. Poly(A)
selected on oligo(dT) cellulose columns.
addition of SDS in the purification steps. Sodium dodecyl sulfate is a
powerful enzyme inhibitor and helps prevent degradation of the RNA by
RNases, but its presence can also inhibit the enzymes required for cDNA
synthesis. If the mRNA intended for use with this kit is suspended in an
SDS solution, the RNA must be phenol extracted and ethanol precipitated.

Ribonucleases A and T1 are widely used in almost all molecular biology
labs and are nearly indestructible. Ribonucleases are produced by microbes
and have also been found in the oils of the skin. Make an effort to use tubes
and micropipet tips which have been handled only with gloves. Use freshly
autoclaved and baked tips and tubes. Usually these precautions are
sufficient, but to be absolutely certain that microcentrifuge tubes and other
components intended for use with RNA are not contaminated, the
components can be treated with DEPC. Diethylpyrocarbonate is extremely
toxic and should be handled with care. Submerge the microcentrifuge tubes
in a 0.1% (v/v) DEPC-treated water solution. Leave the beaker of
submerged tubes in a fume hood overnight and then dispose of the DEPC­treated water. Autoclave the microcentrifuge tubes for at least 30 minutes.
Even though the tubes may still have a sweet DEPC odor, the DEPC is
completely inactivated by this procedure. Place the tubes in a drying oven
overnight. Equipment which cannot be treated by DEPC can be rinsed in a
freshly mixed 3% (v/v) hydrogen peroxide solution, followed by a methanol
rinse. Remember, once the RNA is converted to first-strand cDNA, RNases
are no longer a concern. Caution should still be exercised in maintaining a
sterile, DNase-free environment.

2

is strongly recommended for total

5

Some protocols call for the

+

RNA is

+

RNA Quantification

RNA can be quantified by measuring the optical density of a dilute RNA
solution. The conversion factor for RNA at the wavelength of 260 nm is

40 μg/ml/OD unit as shown in the example below.

Two microliters of a poly(A)
(e.g., OD

= 0.1). Therefore,

260

500

0.1 OD unit

⎛

dilution factor 40 g of RNA / ml = 1000 g of RNA / ml or 1 g of RNA / l×

⎜
⎝

2

28 pBluescript II XR cDNA Library Construction Kit

+

RNA sample is added to 498 ml of water

⎞

×μ μ μ μ

⎟
⎠

If a sample has significant rRNA contamination, the actual amount of
mRNA available for cDNA conversion will be overestimated by this
procedure.

If the amount of poly(A)

+

RNA is below 1.5 μg/synthesis reaction, the RT

may synthesize unclonable hairpin structures. If the amount of poly(A)
RNA is above 7 μg, the percentage of cDNAs that are full length may

decrease. The pBluescript cDNA library construction kit is optimized for

+

5 μg of poly(A)

RNA, but successful libraries have been generated using

the minimums and maximums described here.

Secondary structure may be a problem with certain RNAs, particularly plant
and tumor mRNAs. These samples can be treated with methylmercury
hydroxide (see Appendix II: Methylmercury Hydroxide Treatment).
Treatment with methylmercury hydroxide requires heating the RNA to
65°C. If the RNA contains even a minute amount of RNase, the RNase
activity will increase by several orders of magnitude with the increased
temperature and significantly degrade the RNA. Treatment with
methylmercury hydroxide is therefore recommended only if the RNA is free
of RNases.

APPENDIX II: METHYLMERCURY HYDROXIDE TREATMENT

+

6

Warning Methylmercury hydroxide is an extremely toxic chemical. Wear

gloves and use with caution in a fume hood.

1. Resuspend the mRNA in 20 μl of DEPC-treated water.

2. Incubate at 65°C for 5 minutes.

3. Cool to room temperature.

4. Add 2 μl of 100 mM CH

HgOH.

3

5. Incubate at room temperature for 1 minute.

6. Add 4 μl of 700 mM β-mercaptoethanol (see Preparation of Media and
Reagents).

7. Incubate at room temperature for 5 minutes.

pBluescript II XR cDNA Library Construction Kit 29

APPENDIX III: ALKALINE AGAROSE GELS

Alkaline agarose gels cause DNA to denature and can be used to identify the
presence of a secondary structure called hairpinning. Hairpinning can occur
in either the first- or second-strand reactions when the newly polymerized
strand "snaps back" on itself and forms an antiparallel double helix.

Denaturing gels such as alkaline agarose gels can reveal this secondary
structure and can demonstrate the size range of the first- and second-strand
cDNA.

Note The test cDNA sample will run as a tight band at 1.8 kb and will

show distinctly different intensity between the first and second
strands. This is due to the relative ratio of

amount of dNTP in the first- or second-strand reaction. Normally
the second-strand band will be 1/10 to 1/20 the intensity of the
first-strand band.

Alkaline agarose gels differ from conventional gels in the following ways:

♦ The absence of any buffering capacity in the "buffer" reduces the speed

at which the sample can be run.

♦ The thickness of the typical undried agarose gel causes the radioactive

emissions to be scattered to a degree that makes a clear autoradiograph
difficult to interpret.

The following alternative methods help avoid these complications.

α

-32P dNTP to the

The Slide Technique

The easiest and least expensive method is to use a 5- × 7.5-cm glass slide,
position a minigel comb over it with high tension clips, and add 10 ml of
molten alkaline agarose near the upper center of the slide. The surface
tension of the solution will prevent overflow and produce a small, thin gel
that can be exposed without further drying. Do not allow the teeth of the
comb to overlap the edge of the plate or the surface tension may be broken.
To improve the resolution, pat the gel dry with several changes of Whatman

MM paper after electrophoresis is complete.

3

To prevent radioactive contamination of film cassettes, seal the wet gels in
airtight hybridization bags. Be careful not to trap any air in the hybridization
bag, which could lift the film away from the gel and cause blurring.

30 pBluescript II XR cDNA Library Construction Kit

The Vertical Alkaline Agarose Technique

Vertical alkaline agarose gels can be produced using a vertical gel apparatus
with 1.5-mm spacers. Since the alkaline agarose gels do not have sufficient
friction to remain bound to ordinary glass, a frosted glass plate or gel bond
must be used with the vertical apparatus. The combs normally used for
acrylamide can be used with this apparatus, if the outside teeth are wrapped
in tape to prevent the comb from sinking more than 1.2 cm into the agarose.
The 55°C agarose will solidify almost immediately on contact with the cold
glass plates, so it is essential to load the mold rapidly with a 60-ml syringe.
The comb should already be in the mold, and if it is necessary to reposition
the comb, do it immediately after the gel is poured. In order to reduce the
possibility of destroying the wells when pulling out the comb, place the
solidified gel in a –20°C freezer for 5 minutes immediately prior to
removing the comb. When pulling out the comb, it is essential to avoid a
vacuum between the teeth and the well. Vacuum can be detected when the
well distorts from its normal square shape. When a vacuum occurs, push the
comb to separate the glass plates and break the vacuum. After the samples
have been run and the glass plates are ready to be opened, slide the
unfrosted glass plate off of the alkaline agarose gel instead of prying the
plate away from the gel. Pat the gel dry several times using several pieces of
Whatman 3

Note To prevent radioactive contamination of film cassettes, seal the

MM paper.

wet gels in airtight hybridization bags. Be careful not to trap any
air in the hybridization bag, which could lift the film away from
the gel and cause blurring.

Conventional Submerged Gels

These gels will require drying either by blotting or through the use of a gel
dryer.

Caution Even when multiple layers of absorbent paper are placed

under the gel, free nucleotides can easily contaminate the
drying apparatus. These gels should be poured as thin as
possible and should be dried without heat, if time permits,
and should never be dried above 40°C.

pBluescript II XR cDNA Library Construction Kit 31

Protocol

The following formula makes 80 ml of 1% (w/v) alkaline agarose for
cDNAs in the 1- to 3-kb size range.

Melt 0.8 g of agarose in 72 ml of water. Allow the agarose to cool to 55°C.
During this time, assemble the gel apparatus. Add 8 ml of 10× alkaline

§

buffer

to the cooled agarose, swirl to mix, and pour the agarose
immediately. If buffer is added before the correct temperature is reached, the
agarose may not solidify.

Load the sample in an equal volume of alkaline agarose 2× loading buffer.

§

Run the gel with 1× alkaline buffer at 100 mA and monitor the system for
heat. If the apparatus becomes warmer than 37°C, the amperage should be
reduced. The migration of the BPB in alkaline agarose is similar to the
migration in regular agarose and should be run to at least one-half or three­quarters distance of the gel.

Note The alkali condition causes the blue dye to fade.

§

See Preparation of Media and Reagents.

32 pBluescript II XR cDNA Library Construction Kit

APPENDIX IV: ETHIDIUM BROMIDE PLATE ASSAY—
Q

UANTITATION OF DNA

An accurate quantitation of DNA can be obtained by UV visualization of
samples spotted on EtBr agarose plates. DNA samples of known
concentration are prepared for use as comparative standards in this assay.

Preparation of Ethidium Bromide Plates

Note Prepare the EtBr plates under a fume hood.

Prepare 100 ml of a 0.8% (w/v) agarose and Tris-acetate media. Cool the
molten agarose to 50°C and then add 10 μl of EtBr stock solution

(10 mg/ml). The EtBr stock solution is prepared in dH
dark at 4°C. Swirl to mix the EtBr stock solution and pour the solution into
100-mm petri dishes using ~10 ml/plate. Allow the plates to harden and
incubate the plates at 37°C to dry, if necessary. These plates may be stored
in the dark at 4°C for up to 1 month.

Preparation of Standards

Using a DNA sample of known concentration, make seven serial dilutions in
100 mM EDTA to cover the range from 200 to 10 ng/μl. These standards

may be stored at –20°C for 3 months.

O and is stored in the

2

Plate Assay for Determination of DNA Concentration

Using a marker, label the petri dish to indicate where the sample and the
standards (200, 150, 100, 75, 50, 25, and 10 ng/μl) will be spotted.

Thaw the standards and carefully spot 0.5 μl of each standard onto the
surface of a prepared EtBr plate. Be careful not to dig into the surface of the
plate. Let capillary action pull the small volume from the pipet tip to the
plate surface and do not allow a bubble to form. Change pipet tips between
each standard.

After spotting all of the standards, immediately spot 0.5 μl of the cDNA
sample onto the plate adjacent to the line of standards. Allow all spots to
absorb into the plate for 10–15 minutes at room temperature. Remove the lid
and photograph the plate using a UV lightbox. Compare the spotted sample
of unknown concentration with the standards.

Do not reuse the plates.

Standards and unknowns must be spotted within 10 minutes of each other.

pBluescript II XR cDNA Library Construction Kit 33

TROUBLESHOOTING

Observations Suggestions

Poor first-strand synthesis Always mix and spin the enzymes in a microcentrifuge immediately before use.

Vortex the buffers vigorously until no precipitate is visible.

Ensure that AccuScript RT activity is not inhibited. Minute amounts of SDS or

lithium in the RNA will inhibit the first-strand synthesis reaction. Do not use these in
the RNA preparations. Multiple phenol–chloroform extractions will sometimes
remove the inhibitors.

Ensure that quantity of mRNA is sufficient. Optical density readings may be

obscured by contaminating rRNA or DNA and may give a false indication of the
amount of mRNA used in the synthesis. Repeat the mRNA preparation.

Ensure that [α-32P]dNTP is not degraded or contaminated. If [α-32P]dNTP is

heated, is left at room temperature too long, or is contaminated, it may not
incorporate into cDNA, giving a false indication that synthesis is not occurring.

Poor second-strand synthesis Ensure that the gel results are interpreted correctly. Control RNA will show

distinctly different intensity between the first and second strand. This is due to the
relative amounts of
reaction. Normally, the second strand band will have only 1/10 to 1/20 the
intensity of the first-strand band.

No first-strand synthesis, but good
second-strand synthesis

Hairpinning Ensure that the incubation temperatures are not higher than 16°C. Add second-

Ensure that quantity of mRNA is sufficient. Optical density readings may be

Some sources of RNA may have secondary structure (e.g., tumors, some plants,

Ensure that the quantity of DNA polymerase is not too high. Use a calibrated pipet

Low counts in the drip column
fractions

Poor ligation Ensure that the glycerol concentration is not excessive. Do not use excess ligase.

Always mix and spin the enzymes in a microcentrifuge immediately before use.
Vortex the buffers vigorously until no precipitate is visible.

See the previous suggestions for Poor first-strand synthesis.

Ensure that there is no DNA contamination within the RNA preparation.

strand synthesis reaction components to the first-strand reaction mix on ice and
then transfer the reaction mixture directly to 16°C for incubation. After incubation,
place the samples on ice immediately.

obscured by contaminating rRNA or DNA and may give a false indication of the
amount of mRNA used in the synthesis. Repeat the mRNA preparation.

etc.). The RNA may have to be treated with methylmercury hydroxide to relax the
secondary structure (see Appendix II: Treating with Methylmercury Hydroxide).

to measure the enzyme. Do not submerge the pipet tip completely in the enzyme
solution as additional enzyme will adhere to the outside of the pipet tip.

The number of counts per second per fraction may vary from 0 to 250 cps and
yield primary libraries of >1 × 10
column are from unincorporated [
EtBr plate.

Also, do not submerge the pipet tip completely in the enzyme solution as
additional enzyme will adhere to the outside of the pipet tip.

32

α-

P to the amount of NTP in the first- or second-strand

6

pfu. Most of the counts remaining in the drip

32

α-

P]dNTP. Verify the quantity of cDNA on the

34 pBluescript II XR cDNA Library Construction Kit

PREPARATION OF MEDIA AND REAGENTS

10× Alkaline Buffer (per 50 ml)

3 ml of 5.0 M NaOH
2 ml of 0.5 M EDTA
45 ml of water

LB Agar (per Liter)

10 g of NaCl
10 g of tryptone
5 g of yeast extract
20 g of agar
Adjust pH to 7.0 with 5 N NaOH
Add deionized H

1 liter
Adjust pH to 7.0 with 5 N NaOH
Autoclave
Pour into petri dishes (~25 ml/100-mm

plate)

O to a final volume of

2

2× LB Agarose

450 ml of 2× LB

1.35 g of SeaPrep agarose

Mix on a heated stir plate using a large stir

bar until the agarose is in solution

10× STE Buffer

1 M NaCl
200 mM Tris-HCl (pH 7.5)
100 mM EDTA

LB Broth (per Liter)

10 g of NaCl
10 g of tryptone
5 g of yeast extract
Adjust to pH 7.0 with 5 N NaOH
Add deionized H

1 liter

Adjust to pH 7.0 with 5 N NaOH
Autoclave

O to a final volume of

2

2× LB–Glycerol [12.5% (v/v)]

175 ml of 2× LB broth
25 ml of glycerol [100% (v/v)]

Filter sterilize and store for up to 2 months

at room temperature

2× LB Broth (per Liter)

20 g of NaCl
20 g of tryptone
10 g of yeast extract
Add deionized H

1 liter
Adjust to pH 7.0 with 5 N NaOH
Autoclave

O to a final volume of

2

LB–Ampicillin Broth (per Liter)

1 liter of LB broth, autoclaved
Cool to 55°C
Add 10 ml of 10-mg/ml filter-sterilized

ampicillin

LB–Kanamycin Broth (per Liter)

Prepare 1 liter of LB broth
Autoclave
Cool to 55°C
Add 5 ml of 10-mg/ml-filter-sterilized

kanamycin

LB–Ampicillin Agar (per Liter)

1 liter of LB agar, autoclaved
Cool to 55°C
Add 10 ml of 10-mg/ml filter-sterilized

ampicillin

Pour into petri dishes

(~25 ml/100-mm plate)

LB–Kanamycin Agar (per Liter)

Prepare 1 liter of LB agar
Autoclave
Cool to 55°C
Add 5 ml of 10-mg/ml-filter-sterilized

kanamycin

Pour into petri dishes (~25 ml/100-mm

plate)

pBluescript II XR cDNA Library Construction Kit 35

10× MOPS Buffer

200 mM 3-[N-morpholino]propane-sulfonic

acid (MOPS)
50 mM sodium acetate
10 mM EDTA
Adjust to a final pH of 6.5–7.0 with NaOH
Do not autoclave

Alkaline Agarose 2× Loading
Buffer

200 μl of glycerol
750 μl of water
46 μl of saturated BPB

ll

5 μl of 5 M NaOH

10× Ligase Buffer

500 mM Tris-HCl (pH 7.5)
70 mM MgCl
10 mM DTT

Note rATP is added separately in the

2

ligation reaction

Column-Loading Dye

50% (v/v) glycerol
10% (v/v) 10× STE buffer
40% (w/v) saturated BPB

ll

TE Buffer

10 mM Tris-HCl (pH 7.5)

1 mM EDTA

Formaldehyde Gel Loading Buffer

720 μl of formamide
160 μl of 10× MOPS buffer
260 μl of 37% formaldehyde
100 μl of sterile water
100 μl of EtBr (10 mg/ml)
80 μl of sterile glycerol
80 μl of saturated BPB

Note The loading buffer is not stable

and should be made fresh on the
day of use

ll

in sterile water

NZY+ Broth (per Liter)

10 g of NZ amine (casein hydrolysate)
5 g of yeast extract

Nondenaturing Acrylamide Gel
(5%)

Mix the following in a vacuum flask

5 g of NaCl
Add deionized H

O to a final volume

2

of 1 liter
Adjust to pH 7.5 using NaOH
Autoclave
Add the following filer-sterilized

supplements prior to use:

12.5 ml of 1 M MgCl

12.5 ml of 1 M MgSO

2

4

20 ml of 20% (w/v) glucose (or 10 ml

5 ml of 10× TBE buffer

8.33 ml of a 29:1 acrylamide–bis­ acrylamide solution

36.67 ml of sterile deionized H

O

2

De-gas this mixture under vacuum for

several minutes

Add the following reagents
25 μl of TEMED
250 μl of 10% ammonium persulfate

of 2 M glucose)

ll

To make saturated BPB, add a small amount of bromophenol blue crystals to water and vortex. Centrifuge the sample
briefly and look for the presence of an orange pellet. If a pellet is seen, the solution is saturated. If not, add more crystals
and repeat the procedure.

36 pBluescript II XR cDNA Library Construction Kit

REFERENCES

1. Short, J. M. and Sorge, J. A. (1992) Methods Enzymol 216:495–508.

2. Chomczynski, P. and Sacchi, N. (1987) Anal Biochem 162(1):156–9.

3. Kreiegler, M. (1990). In Gene Transfer and Expression: A Laboratory Manual,
pp. 131–132. Stockton Press, New York.

4. Hanahan, D., Jessee, J. and Bloom, F. R. (1991)

5. Krug, M. S. and Berger, S. L. (1987) Methods Enzymol 152:316–25.

6. Payvar, F. and Schimke, R. T. (1979) J Biol Chem 254(16):7636–42.

Methods Enzymol 204:63–113.

ENDNOTES

B-D®, Falcon®, Luer Lok®, and PrecisionGlide® are registered trademarks of Becton

Dickinson and Company.
GenBank
Parafilm
Sepharose
Styrofoam

®

is a registered trademark of the National Institutes of Health.

®

is a registered trademark of American Can Company.

®

is a registered trademark of Pharmacia Biotech AB.

®

is a registered trademark of Dow Chemical Co.

MSDS INFORMATION

The Material Safety Data Sheet (MSDS) information for Stratagene products is provided on the web at
http://www.stratagene.com/MSDS/. Simply enter the catalog number to retrieve any associated MSDS’s
in a print-ready format. MSDS documents are not included with product shipments.

pBluescript II XR cDNA Library Construction Kit 37