Sutter Instrument ZAP Instruction Manual

Lambda ZAP Premade Library

INSTRUCTION MANUAL

Revision A

BN #935202-12

For In Vitro Use Only

935202-12

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Lambda ZAP Premade Library

CONTENTS

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

Storage Conditions.............................................................................................................................. 1

Additional Materials Required .......................................................................................................... 1

Notice to Purchaser ............................................................................................................................. 1

Introduction......................................................................................................................................... 2

Overview of the Lambda ZAP Vector System...................................................................... 2

Lambda ZAP Vector Map ..................................................................................................... 2

pBluescript SK(-) Vector Map .............................................................................................. 3

Bacterial Host Strains ......................................................................................................................... 4

Host Strain Genotypes...........................................................................................................4

BB4 and XL1-Blue MRF’ Bacterial Strain Descriptions...................................................... 4

Recommended Media............................................................................................................ 5

Establishing an Agar Plate Bacterial Stock ........................................................................... 5

Preparing a –80°C Bacterial Glycerol Stock ......................................................................... 6

Growth of Cells for Plating Phage......................................................................................... 6

Determining Background by Color Selection with IPTG and X-gal..................................... 6

Helper Phage ....................................................................................................................................... 7

Storing the Helper Phage....................................................................................................... 7

Titering the Helper Phage...................................................................................................... 7

Amplifying the Helper Phage................................................................................................ 8

Titering the Library............................................................................................................................ 9

Preparing the Host Bacteria................................................................................................... 9

Titering Protocol.................................................................................................................. 10

Performing Plaque Lifts ................................................................................................................... 11

Hybridizing and Screening............................................................................................................... 12

Antibody Screening Protocol ........................................................................................................... 12

In Vivo Excision of the pBluescript Phagemid from the Lambda ZAP Vector........................... 13

Single-Clone Excision Protocol .......................................................................................... 14

Mass Excision Protocol ....................................................................................................... 16

Appendix: Recovery of Single-Stranded DNA from Cells Containing pBluescript Phagemids 18

Single-Stranded Rescue Protocol ........................................................................................ 19

Troubleshooting ................................................................................................................................ 20

Preparation of Media And Reagents ...............................................................................................20

References .......................................................................................................................................... 22

Endnotes............................................................................................................................................. 22

MSDS Information............................................................................................................................ 22

Lambda ZAP Premade Library

ATERIALS PROVIDED

M

Material provided Quantity

Amplified premade library constructed in the Lambda ZAP vectora 1 ml

Host strainsb

XL1-Blue MRF´ strain 0.5-ml bacterial glycerol stock

BB4 strain 0.5-ml bacterial glycerol stock

f1 helper phagec

R408 interference-resistant helper phage 1 ml

VCSM13 interference-resistant helper phage 1 ml

a

Shipped as a liquid in 7% (v/v) DMSO. On arrival, store the library at –80°C. Do not pass through more than two freeze–

thaw cycles.

b

Use the XL1-Blue MRF´ strain for plating excised phagemids and the BB4 strain for all other manipulations. For host strain

storage conditions, see Bacterial Host Strains.

c

Retiter after 1 month. (Take care not to contaminate the Lambda ZAP vector with this high-titer filamentous helper phage.)
Store at –80°C. We recommend VCSM13 interference-resistant helper phage for single-stranded rescue (see Appendix:
Recovery of Single-Stranded DNA from Cells Containing pBluescript Phagemids).

STORAGE CONDITIONS

All Components: –80°C

ADDITIONAL MATERIALS REQUIRED

14-ml BD Falcon polypropylene round-bottom tubes (BD Biosciences Catalog #352059)

NOTICE TO PURCHASER

The Lambda ZAP vector is covered by Agilent's United States Patent No. 5,128,256. The purchase
of this vector includes a limited, nonexclusive license under such patent rights to use the vector for
the cloning, expression and characterization of genes. This license does not grant rights to (1) use the
Lambda ZAP vector for the reproduction, amplification or modification of the vector;
(2) offer the Lambda ZAP vector or any derivative thereof for resale; (3) distribute or transfer the
Lambda ZAP vector or any derivative thereof to any third party; or (4) incorporate the Lambda ZAP
vector or any derivative thereof in any genomic or cDNA library for resale, distribution or transfer to
any third party. No other license, express, implied or by estoppel, is granted. For information
concerning the availability of licenses to reproduce and/or modify the Lambda ZAP vector, please
contact the Stratagene Technical Services Department at 1-800-894-1304.

Revision A © Agilent Technologies, Inc. 2009.

Lambda ZAP Premade Library 1

INTRODUCTION

Overview of the Lambda ZAP Vector System

The Lambda ZAP vector
library construction and the convenience of a plasmid system (see Figure 1).

The Lambda ZAP vector has six unique cloning sites that will accommodate
DNA inserts from <1 to 10 kb in length. Clones in the Lambda ZAP vector
can be screened with either DNA probes or antibody probes. The
Lambda ZAP vector allows rapid in vivo excision of the
pBluescript phagemid, permitting your insert to be characterized in a
plasmid system (see Figure 2). The polylinker of the pBluescript phagemid
has 21 unique cloning sites flanked by T3 and T7 promoters and a choice of
six different primer sites for DNA sequencing. The phagemid has the
bacteriophage f1 origin of replication, allowing rescue of single-stranded
DNA, which can be used for DNA sequencing or site-directed mutagenesis.
Unidirectional deletions can be made with exonuclease III and mung bean
nuclease by taking advantage of the unique positioning of
5´ and 3´ restriction sites. Transcripts made from the T3 and T7 promoters
generate riboprobes useful in Southern and northern blotting, and the
lacZ promoter may be used to drive expression of fusion proteins suitable
for western blot analysis or protein purification.

The pBluescript phagemid in the Lambda ZAP vector contains the
N-terminus of the lacZ gene, which can be α-complemented by the specific

host strain used. There are 36 amino acids between the MET sequence and
the EcoR I site. A total of 131 amino acids are coded for, but this is
interrupted by the large polylinker.

1, 2

system combines the high efficiency of lambda

Lambda ZAP Vector Map

FIGURE 1 Map of the Lambda ZAP insertion vector.

2 Lambda ZAP Premade Library

pBluescript SK(-) Vector Map

n

β

ampicillin

pBluescript SK-

3.0 kb

pUC ori

pBluescript SK (–) Multiple Cloning Site Regio
(sequence shown 601–826)

T7 Promoter

TTGTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGACGGT...

M13 –20 primer binding site

Bsp106 I

Cla I BamH I

...ATCGATAAGCTTGATATCGAATTCCTGCAGCCCGGGGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCA...

EcoR IEcoR V

T7 primer binding site

Spe ISma I Xba IPst IHind III

SK primer binding site...KS primer binding site

f1 (-) ori

Kpn I

Apa I
EcoO109 I
Dra II

Not I

Eag I

lacZ'

MCS

P lac

BstX I

Kpn I

Sac I

Xho I

KS primer binding site...

Sac II

Hinc II

Acc I

Sal I

Sac I

α

T3 Promoter

...GCTTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCTTGGCGTAATCATGGTCATAGCTGTTTCC

T3 primer binding site

-gal -fragment

M13 Reverse primer binding site

Feature Nucleotide Position

f1 (–) origin of ss-DNA replication 24–330
β-galactosidase α-fragment coding sequence (lacZ’) 463–816

T7 promoter transcription initiation site 643

multiple cloning site 653–760

T3 promoter transcription initiation site 774

lac promoter 817–938

pUC origin of replication 1158–1825

ampicillin resistance (bla) ORF 1976–2833

FIGURE 2 Circular map and polylinker sequence of the pBluescript SK(–) phagemid. The complete sequence and list of
restriction sites are available from www.stratagene.com or from the GenBank

®

database (#X52324).

Lambda ZAP Premade Library 3

BACTERIAL HOST STRAINS

Host Strain Genotypes

Host strain Genotype

BB4 strain LE392.23 [F´lacIqZΔM15 proAB Tn10 (Tetr)
XL1-Blue MRF´ strain Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1

recA1 gyrA96 relA1 lac [F´ proAB lacI

BB4 and XL1-Blue MRF’ Bacterial Strain Descriptions

Two Escherichia coli host strains are supplied with the
Lambda ZAP library: the XL1-Blue
BB4 strain is RecA
Lambda ZAP phage. The RecA
supplied for those hosts requiring a host recombination minus strain. Twelve
hours of growth are usually required before plaques are visible on
XL1-Blue MRF´ cells since XL1-Blue MRF´ supplies the supE
tRNA suppresser, which only weakly suppresses the s100 mutation in the
Lambda ZAP vector. We recommend plating and screening the library on
the BB4 strain. In addition, use of the correct host strain is important when
working with the Lambda ZAP vector as the F´ episome present serves three
purposes.

First, the ΔM15 lacZ gene present on the F´ episome is required for the
β-galactosidase-based nonrecombinant selection strategy. When cDNA is
present in the polylinker, expression from the lacZ gene is disrupted and

white plaques are produced. In contrast, without insert in the polylinker, the
amino terminus of β-galactosidase is expressed and nonrecombinants can be

scored visually by the presence of blue plaques. To produce an
enzymatically active β-galactosidase protein, two domains are required: the
α-region expressed by the vector and the ΔM15 lacZ domain expressed by

the F´ episome. These two domains fold to form a functional protein, the

α-region complementing the missing amino acids resulting from the
ΔM15 mutation. Therefore, in order to utilize the nonrecombinant selection

strategy, the correct host strain must be used to produce a functional
β-galactosidase protein.

Second, the F´ episome expresses the genes forming the F´ pili found on the
surface of the bacteria. Without pili formation, filamentous phage (i.e., M13
or f1) infection could not occur. Because the conversion of a recombinant
Lambda ZAP clone to a pBluescript phagemid requires superinfection with
a filamentous helper phage, the F´ episome is required for in vivo excision
(see In Vivo Excision of the pBluescript Phagemid from the Lambda ZAP
Vector).

+

, which supports vigorous growth of the

–

E. coli host strain XL1-Blue MRF´ is also

q

ZΔM15 Tn10 (Tetr)]

3

MRF´, and BB4 strains. The

4 Lambda ZAP Premade Library

Third, the F´ episome contains the lac repressor (lacIq gene), which blocks
transcription from the lacZ promoter in the absence of the inducer
isopropyl-1-thio-β-
for controlling expression of fusion proteins which may be toxic to the
E. coli. Because the presence of the lacI
can potentially increase the representation or completeness of the library,
BB4 strain is useful for screening the amplified library.

Note The strains used for the Lambda gt11 vector (i.e., Y1088, Y1089,

Recommended Media

D-galactopyranoside (IPTG). This repressor is important

q

repressor in the E. coli host strain

and Y1090) are not suitable for use with the Lambda ZAP vector
because these strains contain the plasmid pMC9, a pBR322
derivative, which contains many of the same sequences as those
found in the phagemid portion of the Lambda ZAP vector. Using
these strains with the Lambda ZAP vector could result in
recombination between the homologous sequences.

Host strain

BB4 strain LB–tetracyclinea LB broth with supplements

XL1-Blue MRF´ strain LB–tetracyclinea LB broth with supplements

a

See Preparation of Media and Reagents.

b

LB broth with 0.2% (w/v) maltose and 10 mM MgSO4.

c

Maltose and magnesium supplements are required for optimal lambda phage receptor expression on the surface of the
bacterial host cell. The media supplements are not required for helper phage infection, but are included in both protocols
for simplified media preparation.

Agar plates and
liquid medium for
bacterial streak
and glycerol stock

Liquid medium for
bacterial cultures prior to
phage attachment

a-c

a-c

Agar plates
and top agar
for plaque
formation

NZYa —

— LB–ampicillina

Agar plates for
excision protocol

Establishing an Agar Plate Bacterial Stock

The bacterial host strains are shipped as bacterial glycerol stocks. On arrival,
prepare the following plates from the bacterial glycerol stocks.

Note The host strains may thaw during shipment. The vials should be

stored immediately at –20° or –80°C, but most strains remain
viable longer if stored at –80°C. It is best to avoid repeated
thawing of the host strains in order to maintain extended viability.

1. Revive the stored cells by scraping off splinters of solid ice with a
sterile wire loop.

2. Streak the splinters onto an LB agar plate containing the appropriate
antibiotic (see Recommended Media), if one is necessary.

3. Incubate the plate overnight at 37°C.

4. Seal the plate with Parafilm

®

laboratory film and store the plate at 4°C

for up to 1 week.

5. Restreak the cells onto a fresh plate every week.

Lambda ZAP Premade Library 5

Preparing a –80°C Bacterial Glycerol Stock

1. In a sterile 50-ml conical tube, inoculate 10 ml of LB broth with the
appropriate antibiotic (see Recommended Media) with one colony from
the plate. Grow the cells to late log phase.

2. Add 4.5 ml of a sterile glycerol-liquid medium solution (prepared by
mixing 5 ml of glycerol + 5 ml of the appropriate medium) to the
bacterial culture from step 1. Mix well.

3. Aliquot into sterile centrifuge tubes (1 ml/tube).

This preparation may be stored at –20°C for 1–2 years or at –80°C for more
than 2 years.

Growth of Cells for Plating Phage

Bacterial cultures for plating phage should be started from a fresh plate
using a single colony and should be grown overnight with vigorous shaking
at 30°C in 50 ml of LB broth supplemented with 0.2% (w/v) maltose and
10 mM MgSO
The lower temperature ensures that the cells will not overgrow. The cells
should be spun at 1000 × g for 10 minutes then gently resuspended in 10 ml
of 10 mM MgSO
10 mM MgSO
phage manipulations described within the manual. Highest efficiencies are
obtained from freshly prepared cells.

. (Do not use tetracycline in the presence of magnesium.)

4

. Before use, dilute cells to an OD

4

. Bacterial cells prepared in this manner can be used for all

4

of 0.5 with

600

Determining Background by Color Selection with IPTG and X-gal

The color selection by α-complementation with the Lambda ZAP vector
requires higher amounts of IPTG and X-gal for generation of the blue color.
Transcription and translation of the fusion protein are normal, but the large
polylinker present within the pBluescript phagemid, which is present in the
Lambda ZAP vector, is partly responsible for the reduced activity of the

β-galactosidase protein—not the promoter. As would be expected, the copy
number of the Lambda ZAP vector is much less per cell than the copy
number of pBluescript phagemids. However, it is important to note that the
color assay is used only for determining the ratio of recombinants to
nonrecombinants within a newly constructed library and is not used for any
other manipulations.

6 Lambda ZAP Premade Library

HELPER PHAGE

(

Two different helper phages are provided with the Lambda ZAP library:
(1) the R408 interference-resistant helper phage and (2) the VCSM13 helper
phage. The R408 helper phage is designed to allow efficient in vivo excision
of the pBluescript phagemid from the Lambda ZAP vector. The
VCSM13 helper phage is recommended for single-stranded rescue
procedures from the excised pBluescript phagemids (see Appendix:

Recovery of Single-Stranded DNA from Cells Containing pBluescript
Phagemids).

Storing the Helper Phage

The R408 helper phage and the VCSM13 helper phage are supplied in
7% dimethylsulfoxide (DMSO) and should be stored at –80°C. The helper
phage may be stored for short periods of time at –20°C or 4°C. It is
important to titer the helper phage prior to each use. Expect titers of
approximately 10
VCSM13 helper phage. If the titer drops over time, prepare a fresh high-titer
stock of the helper phage as outlined in Amplifying the Helper Phage.

Titering the Helper Phage

1. Transfer a colony of XL1-Blue MRF´ cells into 10 ml of LB broth with

10

pfu/ml for the R408 helper phage or 1011 pfu/ml for the

supplements in a 50-ml conical tube. Incubate the conical tube with
shaking at 37°C until growth reaches an OD

of 1.0.

600

2. Dilute the phage (10
and Reagents) and combine 1 μl of each dilution with 200 μl of

XL1-Blue MRF´ cells (OD

3. Incubate the helper phage and the XL1-Blue MRF´ cells for 15 minutes
at 37°C to allow the phage to attach to the cells.

4. Add 3 ml of NZY top agar, melted and cooled to ~48°C, and plate
immediately onto dry, prewarmed NZY agar plates. Allow the plates to
set for 10 minutes.

5. Invert the plates and incubate overnight at 37°C.

Note Helper phage plaques will have a cloudier appearance than

lambda phage plaques.

6. To determine the titer [in plaque-forming units per milliliter (pfu/ml)],
use the following formula:

⎡

Number of plaques pfu dilution factor

⎢
⎣

where the volume plated (in microliters) refers to the volume of the

helper phage solution added to the cells.

–4

–10–7) in SM buffer (See Preparation of Media

= 1.0).

600

×

)

Volume plated l

μ

(

⎤

1000 l / ml

×

)

⎥
⎦

μ

Lambda ZAP Premade Library 7

Amplifying the Helper Phage

1. Transfer a colony of XL1-Blue MRF´ cells into 10 ml of LB broth with
supplements in a 50-ml conical tube. Incubate the conical tube with
shaking at 37°C until growth reaches an OD

of 0.3.

600

Note An OD

of 0.3 corresponds to 2.5 × 108 cells/ml.

600

2. Add the helper phage at a multiplicity of infection (MOI) of 20:1
(phage-to-cells ratio).

3. Incubate the conical tube at 37°C for 15 minutes to allow the phage to
attach to the cells.

4. Incubate the conical tube with shaking at 37°C for 8 hours.

Note When amplifying VCSM13 helper phage, add kanamycin to a

μ

final concentration of 25

g/ml after 30 minutes of growth.

5. Heat the conical tube at 65°C for 15 minutes.

6. Spin down the cell debris and transfer the supernatant to a fresh conical
tube.

10

7. The titer of the supernatant should be between 7.5 × 10

12

1.0 × 10

1.0 × 10

pfu/ml for R408 helper phage or between 1.0 × 1011 and

12

pfu/ml for VCSM13 helper phage.

and

Note Helper phage plaques will have a cloudier appearance than

lambda phage plaques.

8. Add dimethylsulfoxide (DMSO) to a final concentration of 7% (v/v)
and store at –80°C.

9. For further details about helper phage titering or amplification, please
see

Titering the Helper Phage or Reference 4.

8 Lambda ZAP Premade Library

TITERING THE LIBRARY

Preparing the Host Bacteria

1. Streak the BB4 cells onto an LB–tetracycline agar plate. Incubate the

2. Inoculate 50 ml of LB broth with supplements in a sterile flask with a

3. Incubate with shaking at 37°C for 4–6 hours (do not grow past an

4. Pellet the bacteria at 1000 × g for 10 minutes.

plate overnight at 37°C.

single colony of the BB4 host.

Note Do not add antibiotic to the overnight culture or to the

titering plates. The antibiotic will bind to the bacterial cell
wall and will inhibit the ability of the phage to infect the cell.

OD

of 1.0). Alternatively, grow overnight at 30°C, shaking at

600

200 rpm.

Note The lower temperature keeps the bacteria from overgrowing,

thus reducing the number of nonviable cells. Phage can
adhere to nonviable cells resulting in a decreased titer.

5. Gently resuspend the cell pellet in 25 ml sterile 10 mM MgSO

4

Note For later use, store the cells at 4°C overnight in

10 mM MgSO

.

4

.

Lambda ZAP Premade Library 9

Titering Protocol

A background test can be completed by plating several hundred plaques on a
plate [see

X-gal

Determining Background by Color Selection with IPTG and

]. Add 15 μl of 0.5 M IPTG (in water) and 50 μl of 250 mg/ml X-gal

[in dimethylformamide (DMF)] to 2–3 ml of NZY top agar, melted and
cooled to ~48°C. The higher concentrations of IPTG and X-gal used in the
plating often result in the formation of a precipitate, which disappears after
incubation. The IPTG and X-gal should be added separately, with mixing in
between additions, to the NZY top agar to minimize the formation of this
precipitate. Plate immediately on NZY agar plates. Plaques are visible after
incubation for 6–8 hours at 37°C, although color detection requires
overnight incubation. Background plaques are blue, while recombinant
plaques are white.

1. Dilute the BB4 cells (from step 5 of

Titering the Library) to an OD

600

Preparing the Host Bacteria in

of 0.5 with sterile 10 mM MgSO4.

Note The bacteria should be used immediately following dilution.

2. For amplified library titering, first dilute the amplified phage stock in
SM buffer by the following amounts: 1:10,000, 1:100,000, 1:1,000,000.

Add 1 μl of each dilution to 200 μl of host cells at an OD

of 0.5.

600

Note The premade library has been through one round of

amplification.

3. Incubate the phage and the bacteria at 37°C for 15 minutes to allow the
phage to attach to the cells.

4. Add the following components:

2–3 ml of NZY top agar (melted and cooled to ~48°C).
15 μl of 0.5M IPTG (in water)
50 μl of X-gal [250 mg/ml (in DMF)]

5. Plate immediately onto dry, prewarmed NZY agar plates and allow the
plates to set for 10 minutes. Invert the plates and incubate at 37°C.

6. Plaques should be visible after 6–8 hours, although color detection
requires overnight incubation. Background plaques are blue and should

be < 1× 10

5

pfu/μg of arms, while recombinant plaques will be white

(clear) and should be 10–100-fold above the background.

10 Lambda ZAP Premade Library

PERFORMING PLAQUE LIFTS

1. Titer the library suspension to determine the concentration using
BB4 cells.

2. Combine the equivalent of 5 × 10
prepared BB4 cells at an OD

600

4

pfu/plate and 600 μl of freshly

of 0.5.

3. Incubate the bacteria and phage mixture at 37°C for 15 minutes to
allow the phage to attach to the cells.

4. Add 6.5 ml of NZY top agar (~48°C) to the bacteria and phage mixture.

5. Quickly pour the plating culture onto a dry, prewarmed 150-mm
NZY agar plate, which is at least 2 days old. Carefully swirl the plate to
distribute the cells evenly. Allow the plates to set for 10 minutes. (Use

6

20 plates to screen 1 × 10

pfu.)

6. Invert the plates and incubate at 37°C for ~8 hours.

7. Chill the plates for 2 hours at 4°C to prevent the top agar from sticking
to the nitrocellulose membrane.

Note Use forceps and wear gloves for the following steps.

8. Place a nitrocellulose membrane onto each NZY agar plate for
2 minutes to allow the transfer of the phage particles to the membrane.
Use a needle to prick through the membrane and agar for orientation.
(If desired, waterproof ink in a syringe needle may be used.)

Notes If making duplicate nitrocellulose membranes, allow the

second membrane to transfer for ~4 minutes.

®

Pyrex

dishes are convenient for the following steps. All

solutions should be at room temperature.

a. Denature the nitrocellulose-bound DNA after lifting by

submerging the membrane in a 1.5 M NaCl and 0.5 M NaOH
denaturation solution for 2 minutes.

Note If using charged nylon, wash with gloved fingertips to remove

the excess top agar.

b. Neutralize the nitrocellulose membrane for 5 minutes by

submerging the membrane in a 1.5 M NaCl and 0.5 M Tris-HCl
(pH 8.0) neutralization solution.

c. Rinse the nitrocellulose membrane for no more than 30 seconds by

submerging the membrane in a 0.2 M Tris-HCl (pH 7.5) and
2× SSC buffer solution (see

Preparation of Media and Reagents).

Lambda ZAP Premade Library 11

9. Blot briefly on a Whatman® 3MM paper.

10. Crosslink the DNA to the membranes using the autocrosslink setting on
the Stratalinker UV crosslinker* (120,000 μJ of UV energy) for

~30 seconds. Alternatively, oven bake at 80°C for ~1.5–2 hours.

11. Store the stock agar plates of the transfers at 4°C to use after screening.

HYBRIDIZING AND SCREENING

Following the preparation of the membranes for hybridization, perform
prehybridization, probe preparation, hybridization, and washes for either
oligonucleotide probes or double-stranded probes and then expose the
membranes to film as outlined in standard methodology texts.
these procedures, perform secondary and tertiary screenings also as outlined
in the standard methodology texts.
Sambrook

4

et al.

for suggested phage miniprep and maxiprep procedures.

ANTIBODY SCREENING PROTOCOL

A complete manual for immunoscreening is supplied with the Stratagene

picoBlue immunoscreening kit. This kit is available with goat anti-rabbit

and goat anti-mouse antibodies [Catalog #200371 (goat anti-rabbit) and
200372 (goat anti-mouse)].

* Available as Stratagene Catalog #400071 (1800) and #400075 (2400).

4, 5

Following

4, 5

After an isolate is obtained, refer to

12 Lambda ZAP Premade Library

IN VIVO EXCISION OF THE PBLUESCRIPT PHAGEMID FROM THE LAMBDA
ZAP VECTOR

The Lambda ZAP vector is designed to allow simple, efficient in vivo
excision and recircularization of any cloned insert contained within the
lambda vector to form a phagemid containing the cloned insert. This in vivo
excision depends on the placement of the DNA sequences within the lambda
phage genome and on the presence of a variety of proteins, including
f1 bacteriophage-derived proteins. The f1 phage proteins recognize a region
of DNA normally serving as the f1 bacteriophage origin of replication. This
origin of replication can be divided into two overlying parts: (1) the site of
initiation and (2) the site of termination for DNA synthesis.
regions are subcloned separately into the Lambda ZAP vector. The lambda
phage (target) is made accessible to the f1-derived proteins by
simultaneously infecting a strain of
the f1 bacteriophage.

Inside
recognize the initiator DNA that is within the lambda vector. One of these
proteins then nicks one of the two DNA strands. At the site of this nick, new
DNA synthesis begins and duplicates whatever DNA exists in the lambda
vector "downstream" (3´) of the nicking site. DNA synthesis of a new single
strand of DNA continues through the cloned insert until a termination
signal, positioned 3´ to the initiator signal, is encountered within the
constructed lambda vector. The single-stranded DNA molecule is
circularized by the gene II product from the f1 phage, forming a circular
DNA molecule containing the DNA between the initiator and terminator. In
the case of the Lambda ZAP vector, this includes all sequences of the
pBluescript SK(–) phagemid and the insert, if one is present. This
conversion is the "subcloning" step, since all sequences associated with
normal lambda vectors are positioned outside of the initiator and terminator
signals and are not contained within the circularized DNA. In addition, the
circularizing of the DNA automatically recreates a functional f1 origin as
found in f1 bacteriophage or phagemids.

Signals for “packaging” the newly created phagemid are contained within
the f1 terminator origin DNA sequence. They permit the circularized DNA
to be “packaged” and secreted from the
secreted, the
be removed from the supernatant by heating at 70°C. The heat treatment
kills all the
treatment. For production of double-stranded DNA, the “packaged”
pBluescript DNA is mixed with fresh
LB–ampicillin plates to produce colonies. DNA from minipreps of these
colonies can be used for analysis of insert DNA including DNA sequencing,
subcloning, mapping, and expression.

Miniprep DNA is also used for subsequent production of single-stranded
DNA suitable for dideoxy-sequencing and site-specific mutagenesis.

E. coli, the "helper" proteins (i.e., proteins from f1 or M13 phage)

E. coli cells used for in vivo excision of the cloned DNA can

E. coli cells while the phagemid remains resistant to the heat

E. coli with both the lambda vector and

E. coli. Once the phagemid is

E. coli cells and spread on

6

These two

Lambda ZAP Premade Library 13

Single-Clone Excision Protocol

Day 1

1. Core the plaque of interest from the agar plate and transfer the plaque
to a sterile microcentrifuge tube containing 500 μl of SM buffer and
20 μl of chloroform. Vortex the microcentrifuge tube to release the

phage particles into the SM buffer. Incubate the microcentrifuge tube
for 1–2 hours at room temperature or overnight at 4°C. (This phage
stock is stable for up to 6 months at 4°C.)

2. Grow separate 50-ml overnight cultures of BB4 and
XL1-Blue MRF’ cells in LB broth with supplements at 30°C.

Day 2

3. Gently spin down the BB4 and XL1-Blue MRF’ cells (1000 × g).
Resuspend each of the cell pellets in 25 ml of 10 mM MgSO
the OD
cells to an OD

4. Combine the following components in a 14-ml BD Falcon
polypropylene round-botton tube:

of the cell suspensions, then adjust the concentration of the

600

200 μl of BB4 cells at an OD
250 μl of phage stock (containing >1 × 10
1 μl of the R408 helper phage (>1 × 10

of 1.0 (8 × 108 cells/ml) in 10 mM MgSO4.

600

of 1.0

600

5

phage particles)

6

pfu/μl)

. Measure

4

Note Briefly spin the lambda phage stock to ensure that the

chloroform is separated completely before removing the
aliquot used in the excision reaction.

5. Incubate the BD Falcon polypropylene tube at 37°C for 15 minutes to
allow the phage to attach to the cells.

6. Add 3 ml of LB broth with supplements and incubate the BD Falcon
polypropylene tube for 2.5–3 hours at 37°C with shaking. Because
clonal representation is not relevant, single-clone excision reactions can
be safely performed overnight.

Note The turbidity of the media is not indicative of the success of

the excision.

7. Heat the BD Falcon polypropylene tube at 65–70°C for 20 minutes to
lyse the lambda phage particles and the cells. Spin the tube at 1000 ×

g

for 15 minutes to pellet the cell debris.

8. Decant the supernatant into a sterile 14-ml BD Falcon polypropylene
round-bottom tube. This stock contains the excised pBluescript
phagemid packaged as filamentous phage particles. (This stock may be
stored at 4°C for 1–2 months.)

14 Lambda ZAP Premade Library

9. To plate the excised phagemids, add 200 μl of freshly grown
XL1-Blue MRF’ cells from step 3 (OD

= 1.0) to two

600

1.5-ml microcentrifuge tubes. Add 100 μl of the phage supernatant
(from step 8 above) to one microcentrifuge tube and 10 μl of the phage

supernatant to the other microcentrifuge tube.

10. Incubate the microcentrifuge tubes at 37°C for 15 minutes.

11. Plate 200 μl of the cell mixture from each microcentrifuge tube on
LB–ampicillin agar plates (100 μg/ml) and incubate the plates

overnight at 37°C.

Due to the high-efficiency of the excision process, it may be necessary to
dilute the supernatant to achieve single-colony isolation.

Colonies appearing on the plate contain the pBluescript double-stranded
phagemid with the cloned DNA insert.

To maintain the pBluescript phagemid, streak the colony on a new
LB–ampicillin agar plate. For long-term storage, prepare a bacterial glycerol
stock and store at –80°C.

VCSM13 helper phage is recommended for the single-stranded rescue
procedure. The single-stranded rescue procedure can be found in

Appendix:
Recovery of Single-Stranded DNA from Cells Containing
pBluescript Phagemids

Lambda ZAP Premade Library 15

Mass Excision Protocol

Day 1

1. Grow separate 50-ml overnight cultures of BB4 and

XL1-Blue MRF’ cells in LB broth with supplements at 30°C.

Day 2

2. Gently spin down the BB4 and XL1-Blue MRF’ cells (1000 × g).

Resuspend each of the cell pellets in 25 ml of 10 mM MgSO
the OD
cells to an OD

3. In a 50-ml conical tube, combine a portion of the amplified lambda

bacteriophage library with BB4 cells at a MOI of 1:10 lambda phage­to-cell ratio. Excise 10- to 100-fold more lambda phage than the size of
the primary library to ensure statistical representation of the excised
clones. Add R408 helper phage at a 10:1 helper phage-to-cells ratio to
ensure that every cell is co-infected with lambda phage and helper
phage.

For example, use

of the cell suspensions, then adjust the concentration of the

600

of 1.0 (8 × 108 cells/ml) in 10 mM MgSO4.

600

7

pfu of the lambda phage (i.e., 10- to 100-fold above the

10

primary library size)

8

10

BB4 cells (1:10 lambda phage-to-cell ratio, noting that an

OD

of 1.0 corresponds to 8 × 108 cells/ml)

600

9

pfu of R408 helper phage (10:1 helper phage-to-cells ratio)

10

. Measure

4

Note Briefly spin the lambda phage stock to ensure that the

chloroform is separated completely before removing the
aliquot used in the excision reaction.

4. Incubate the conical tube at 37°C for 15 minutes to allow the phage to

attach to the cells.

5. Add 20 ml of LB broth with supplements and incubate the conical tube

for 2.5–3 hours at 37°C with shaking.

Notes Incubation times for mass excision in excess of 3 hours may

alter the clonal representation.

The turbidity of the media is not indicative of the success of

the excision.

6. Heat the conical tube at 65–70°C for 20 minutes to lyse the lambda

phage particles and the cells.

7. Spin the conical tube at 1000 ×

g for 10 minutes to pellet the cell debris

and then decant the supernatant into a sterile conical tube.

16 Lambda ZAP Premade Library

8. To titer the excised phagemids, combine 1 μl of this supernatant with

200 μl of XL1-Blue MRF’ cells from step 2 in a

1.5-ml microcentrifuge tube.

9. Incubate the microcentrifuge tube at 37°C for 15 minutes.

10. Plate 100 μl of the cell mixture onto LB–ampicillin agar plates

(100 μg/ml) and incubate the plates overnight at 37°C.

Note It may be necessary to further dilute the cell mixture to

achieve single-colony isolation.

At this stage, colonies may be selected for plasmid preps, or the cell mixture
may be plated directly onto filters for colony screening.

Lambda ZAP Premade Library 17

APPENDIX: RECOVERY OF SINGLE-STRANDED DNA FROM CELLS
CONTAINING PBLUESCRIPT PHAGEMIDS

pBluescript is a phagemid that can be secreted as single-stranded DNA in
the presence of M13 helper phage. These phagemids contain the intergenic
(IG) region of a filamentous f1 phage. This region encodes all of the

cis-acting functions of the phage required for packaging and replication. In
E. coli with the F

phagemids will be secreted as single-stranded f1 "packaged" phage when the
bacteria has been infected by a helper phage. Since these filamentous helper
phages (M13, f1) will not infect
pili,

it is essential to use XL1-Blue MRF´ or a similar strain containing

the F´ episome.

The Stratagene Products Division offers helper phages that
package pBluescript phagemids. Typically, 30–50 pBluescript molecules are
packaged/helper phage DNA molecule. pBluescript phagemids are offered
with the IG region in either of two orientations: pBluescript (+) is replicated

such that the sense strand of the β-galactosidase gene is secreted within the
phage particles; pBluescript (–) is replicated such that the antisense strand of

the β-galactosidase gene is secreted in the phage particles.

Yields of single-stranded (ss)DNA depend on the specific insert sequence.
For most inserts, over 1 μg of ssDNA can be obtained from a

1.5-ml miniprep if grown in XL1-Blue MRF´. A faint single-strand helper
phage band may appear on a gel at ~4 kb for R408 or at 6 kb for VCSM13.
This DNA mixture can be sequenced with primers that are specific for
pBluescript and do not hybridize to the helper phage genome.

Site-specific mutagenesis is also possible using standard techniques. The
advantages of using pBluescript phagemids for either purpose are as
follows: (1) pBluescript phagemids do not replicate via the M13 cycle,
lessening the tendency to delete DNA inserts, therefore it is unlikely that
even 10-kb inserts will be deleted. (2) "Packaging" of pBluescript
phagemids containing inserts is efficient since the pBluescript vector is
significantly smaller than wild-type M13. (3) Oligonucleotide mutagenesis
in pBluescript vectors is advantageous because the mutagenized insert is
located between the T3 and T7 promoters. The resultant mutant transcripts
can be synthesized

VCSM13 and R408 helper phage produce the largest amount of single­strand pBluescript. R408 (single-strand size ~4 kb) is more stable and can be
grown more easily. VCSM13 (single-strand size ~6 kb), is more efficient at
single-stranded DNA rescue and yields more single-stranded phagemid;
however it is more unstable and reverts to wild-type more frequently. This
difficulty can be addressed by periodically propagating VCSM13 in the
presence of kanamycin. VCSM13 (a derivative of M13KO7) has a
kanamycin gene inserted into the intergenic region, while R408 has a
deletion in that region. We suggest R408 for excision of pBluescript from
the Lambda ZAP vector and VCSM13 for single-stranded rescue.

+

phenotype (containing an F´ episome), pBluescript

E. coli without an F´ episome coding for

7, 8

preferentially

in vitro without further subcloning.

18 Lambda ZAP Premade Library

Single-Stranded Rescue Protocol

1. Inoculate a single colony into 5 ml of 2× YT broth§ containing

100 μg/ml ampicillin and VCSM13 or R408 helper phage at

7

10

–108 pfu/ml (MOI ~10).

2. Grow the culture at 37°C with vigorous aeration for 16–24 hours, or

until growth has reached saturation.

Note If using VCSM13, after 1–2 hours, add kanamycin to

70

μ

g/ml to select for infected cells.

3. Centrifuge 1.5 ml of the cell culture for 5 minutes in a microcentrifuge.

4. Remove 1 ml of the supernatant to a fresh tube, then add 150 μl of a

solution containing 20% PEG8000 and 2.5 M NaCl. Allow phage
particles to precipitate on ice for 15 minutes.

Note For increased yield, perform the PEG precipitation overnight

at 4°C.

5. Centrifuge for 5 minutes in a microcentrifuge. (A pellet should

be obvious.)

6. Remove supernatant. Centrifuge the PEG pellets a few seconds more to

collect residual liquid, then remove and discard the residual liquid.

7. Resuspend the pellet in 400 μl of 0.3 M NaOAc (pH 6.0) and

1 mM EDTA by vortexing vigorously.

8. Extract with 1 volume phenol–chloroform and centrifuge for

1–2 minutes to separate phases.

9. Transfer the aqueous phase to a fresh tube and add 1 ml of ethanol.

Centrifuge for 5 minutes.

10. Remove ethanol and dry the DNA pellet.

11. Dissolve the pellet in 25 μl of TE buffer

12.

Analyze 1–2 μl on an agarose gel.

§

.

§

See Preparation of Media and Reagents.

Lambda ZAP Premade Library 19

TROUBLESHOOTING

Observation Suggestion

The number of colonies is too low

* ABLE competent cells (Stratagene Catalog #200170–200172) and ABLE electroporation competent cells (Stratagene

Catalog #200160–200162) are available separately.

Verify that the titer on the tubes is current and correct and use only calibrated
pipettors. The molar ratios of lambda phage to cells to helper phage is critical

If an excision is unsuccessful, prepare a high-titer stock of the phage and repeat
the excision procedure, as excision efficiencies are directly related to the Lambda
ZAP phage titer

Poor rescue may be a result of toxic cDNA clones which can be isolated in lambda
vectors but not in plasmid vectors. The ABLE C strain* and the ABLE K strain*
reduce the copy number of common cloning vectors by ~4- and 10-fold,
respectively, enhancing the probability that a toxic clone will be propagated.
Positive clones observed on initial screening as lambda plaques can be excised
and introduced into the ABLE strains. Excised phagemid libraries can also be
screened directly in the ABLE strains

The lambda phage stock aliquot used for in vivo excision cannot contain
chloroform, as chloroform lyses the E. coli cells. Briefly spin the lambda phage
stock to ensure that the chloroform is separated completely before removing the
aliquot

PREPARATION OF MEDIA AND REAGENTS

LB Agar (per Liter)

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

O to a final volume of

2

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

LB–Tetracycline Broth (per Liter)

Prepare 1 liter of LB broth
Autoclave
Cool to 55°C
Add 1.25 ml of 10 mg/ml-filter-sterilized

tetracycline
Store broth in a dark, cool place as

tetracycline is light-sensitive

LB–Ampicillin Agar (per Liter)

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

ampicillin

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

LB–Tetracycline Agar (per Liter)

Prepare 1 liter of LB agar
Autoclave
Cool to 55°C
Add 1.25 ml of 10 mg/ml-filter-sterilized

tetracycline
Pour into petri dishes (~25 ml/100-mm plate)
Store plates in a dark, cool place or cover

plates with foil if left out at room

temperature for extended time periods as

tetracycline is light-sensitive

20 Lambda ZAP Premade Library

LB Broth

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

liter
Adjust pH to 7.0 with 5 N NaOH
Autoclave

O to a final volume of 1

2

LB Broth with Supplements

Prepare 1 liter of LB broth
Autoclave
Add the following filter-sterilized

supplements prior to use
10 ml of 1 M MgSO
3 ml of a 2 M maltose solution or 10 ml

of 20% (w/v) maltose

4

NZY Agar (per Liter)

5 g of NaCl
2 g of MgSO
5 g of yeast extract
10 g of NZ amine (casein hydrolysate)
15 g of agar
Add deionized H

1 liter
Adjust the pH to 7.5 with NaOH
Autoclave
Pour into petri dishes (~80 ml/150-mm plate)

.

7H

O

4

2

O to a final volume of

2

NZY Top Agar (per Liter)

Prepare 1 liter of NZY broth
Add 0.7% (w/v) agarose
Autoclave

TE Buffer

5 mM Tris-HCl (pH 7.5)

0.1 mM EDTA

20× SSC Buffer (per Liter)

175.3 g of NaCl

88.2 g of sodium citrate

800.0 ml of deionized H
Adjust to pH 7.0 with a few drops of

10 N NaOH
Add deionized H

O to a final volume of

2

1 liter

O

2

NZY Broth (per Liter)

5 g of NaCl
2 g of MgSO

. 7H2O

4

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

O to a final volume of

2

1 liter
Adjust the pH to 7.5 with NaOH
Autoclave

SM Buffer (per Liter)

5.8 g of NaCl

2.0 g of MgSO

· 7H2O

4

50.0 ml of 1 M Tris-HCl (pH 7.5)

5.0 ml of 2% (w/v) gelatin
Add deionized H

O to a final volume of

2

1 liter
Autoclave

2× YT Broth (per Liter)

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

1 liter
Adjust to pH 7.5 with NaOH
Autoclave

O to a final volume of

2

Lambda ZAP Premade Library 21

REFERENCES

ENDNOTES

1. Short, J. M., Fernandez, J. M., Sorge, J. A. and Huse, W. D. (1988) Nucleic Acids Res

16(15):7583-600.

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

3. Bullock, W. O., Fernandez, J. M. and Short, J. M. (1987) Biotechniques 5(4):376–378.

4. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory
Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

5. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G. et al. (1987).
Current Protocols in Molecular Biology. John Wiley and Sons, New York.

6. Dotto, G. P., Horiuchi, K. and Zinder, N. D. (1984) J Mol Biol 172(4):507-21.

7. Dente, L., Cesareni, G. and Cortese, R. (1983) Nucleic Acids Res 11(6):1645-55.

8. Mead, D. A., Skorupa, E. S. and Kemper, B. (1985) Nucleic Acids Res 13(4):1103-18.

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22 Lambda ZAP Premade Library