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