Sutter Instrument ZAP II Instruction Manual

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Lambda ZAP® II Premade Library

INSTRUCTION MANUAL

Revision A

BN #946306-11

For In Vitro Use Only

946306-11

LIMITED PRODUCT WARRANTY

This warranty limits our liability to replacement of this product. No other warranties of any kind, express or implied, including without limitation, implied warranties of merchantability or fitness for a particular purpose, are provided by Stratagene. Stratagene shall have no liability for any direct, indirect, consequential, or incidental damages arising out of the use, the results of use, or the inability to use this product.

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Lambda ZAP® II Premade Library

CONTENTS

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

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

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

Notice to Purchaser ............................................................................................................................. 2

Introduction......................................................................................................................................... 3

Overview of the Lambda ZAP

Lambda ZAP

pBluescript

II Vector Map................................................................................................ 3

SK(–) Vector Map............................................................................................ 4

cDNA Synthesis ....................................................................................................................5

Bacterial Host Strains ......................................................................................................................... 6

Host Strain Genotypes...........................................................................................................6

XL1-Blue MRF’ Bacterial Strain Description....................................................................... 6

Recommended Media............................................................................................................ 7

II Vector System ................................................................ 3

Establishing an Agar Plate Bacterial Stock ........................................................................... 7

Preparing a –80°C Bacterial Glycerol Stock ......................................................................... 8

Growth of Cells for Plating Phage......................................................................................... 8

Determining Background by Color Selection with IPTG and X-gal..................................... 8

Helper Phage ....................................................................................................................................... 9

Storing the Helper Phage....................................................................................................... 9

Titering the Helper Phage...................................................................................................... 9

Amplifying the Helper Phage.............................................................................................. 10

Titering the Library.......................................................................................................................... 11

Preparing the Host Bacteria................................................................................................. 11

Titering Protocol.................................................................................................................. 11

Amplifying the Library..................................................................................................................... 13

Performing Plaque Lifts ................................................................................................................... 15

Hybridizing and Screening............................................................................................................... 16

Antibody Screening Protocol ........................................................................................................... 16

In Vivo Excision of the pBluescript

SK(–) Phagemid from the Lambda ZAP II Vector.......... 17

In Vivo Excision Protocols Using the ExAssist® Interference-Resistant Helper Phage with

SOLR™ Strain ..........................................................................................................................18

Single-Clone Excision Protocol .......................................................................................... 18

Mass Excision Protocol ....................................................................................................... 20

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

Phagemids22

Single-Stranded Rescue Protocol ........................................................................................ 23

Troubleshooting ................................................................................................................................ 24

Preparation of Media and Reagents................................................................................................ 25

References .......................................................................................................................................... 27

Endnotes............................................................................................................................................. 27

MSDS Information............................................................................................................................ 27

Lambda ZAP® II Premade Library

ATERIALS PROVIDED

Materials provided Quantity

Amplified premade library constructed in the Lambda ZAP® II vectora 1 ml

Host strainsb

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

SOLR™ strain 0.5-ml bacterial glycerol stock

f1 helper phagec

ExAssist® interference-resistant helper phage 1 ml

VCSM13 interference-resistant helper phage 1 ml

Premade libraries have been amplified one time and frozen in the presence of 7% DMSO. Upon arrival, store at –80°C.

Do not pass through more than two freeze–thaw cycles.

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

strain storage conditions, see Bacterial Host Strains.

Retiter after 1 month. (Take care not to contaminate the Lambda ZAP II vector with this high-titer filamentous helper phage.) Store at –80°C. Stratagene recommends VCSM13 interference-resistant helper phage for single-stranded rescue. The ExAssist interference-resistant helper phage is recommended for excision of the pBluescript phagemid from the Lambda ZAP II vector. It should not be used for single-stranded rescue in general, because this f1 helper phage possesses α-complementing β-galactosidase sequences which may interfere with sequencing or site-directed mutagenesis where oligonucleotide primers hybridize to β-galactosidase sequences (e.g., M13–20 primer).

STORAGE CONDITIONS

Premade Library: –80°C Bacterial Glycerol Stocks: –80°C Helper Phage: –80°C

ADDITIONAL MATERIALS REQUIRED

Isopropyl-1-thio-β-D-galactopyranoside (IPTG) 5-Bromo-4-chloro-3-indoyl-β-

D-galactopyranoside (X-gal)

Lambda ZAP® II Premade Library 1

NOTICE TO PURCHASER

The Lambda ZAP® II vector is covered by Stratagene'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 II vector for the reproduction, amplification or modification of the vector; (2) offer the Lambda ZAP II vector or any derivative thereof for resale; (3) distribute or transfer the Lambda ZAP II vector or any derivative thereof to any third party; or (4) incorporate the Lambda ZAP II 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 II vector, please contact Stratagene's Technical Services Department at 1-800-424-5444.

2 Lambda ZAP® II Premade Library

INTRODUCTION

Overview of the Lambda ZAP® II Vector System

The Lambda ZAP® II system combines the high efficiency of lambda library construction and the convenience of a plasmid system with improved blue– white color selection (see Figure 1). The original Lambda ZAP vector contains the Sam100 mutation, thus limiting the choice of suitable host strains to those containing a supF genotype. Lambda ZAP II, a new variation of the Lambda ZAP vector, no longer contains the Sam100 mutation, therefore, highly efficient growth can be obtained on many nonsupF strains, including XL1-Blue MRF´ cells. Use of the XL1-Blue MRF´ host strain with Lambda ZAP II enhances the blue color produced by nonrecombinant phage, thereby improving blue–white color selection.

As with the original Lambda ZAP vector, the Lambda ZAP II vector has six unique cloning sites that will accommodate DNA inserts from 0 to 10 kb in length. Clones in the Lambda ZAP II vector can be screened with either DNA probes or antibody probes and allows in vivo rapid excision of the pBluescript plasmid system (see Figure 2). derived from pUC19, has 21 unique cloning sites flanked by T3 and T7 promoters and a choice of 6 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 sitedirected 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 SK(–) phagemid in the Lambda ZAP II 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 from the MET sequence to the EcoR I site. A total of 131 amino acids are coded for, but this is interrupted by the large polylinker.

phagemid, allowing your insert to be characterized in a

The polylinker of the pBluescript phagemid,

Lambda ZAP® II Vector Map

Lambda ZAP® II Premade Library 3

Left End 0 kb

Mlu I 0.46

Bgl II 0.42

Pvu I 11.93

Mlu I 5.5

Apa I 10.09

SnaB I 12.19

A - J

FIGURE 1 Map of the Lambda ZAP® II insertion vector.

Mlu I 17.79

Mlu I 15.37

Kpn I 17.05

Kpn I 18.56

MCS

pBluescript®

Hind III 22.48

Sac I 22.03

BamH I 22.97

Kpn I 22.14

Sma I 26.62

att int xis c1857 (nin5)

BamH I 29.50

Bgl II 30.71

Hind III 31.90

Hind III 32.47

Bgl II 33.10

Bgl II 33.75

Bgl II 33.81

Sma I 34.89

Hind III 36.46

Nar I 38.00

Right End 40.82 kb

pBluescript® SK(–) Vector Map

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

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).

4 Lambda ZAP® II Premade Library

cDNA Synthesis

Premade Lambda Zap II libraries are primed with an oligo poly(dt) linkerprimer, digested to create blunt ends, and ligated with EcoR I adaptors. The adaptors are comprised of 9- and 13-mer oligonucleotides, which are complementary to each other and have an EcoR I cohesive end. The adaptors have the following sequence:

5´ AATTCGGCACGAG 3´ 3´ GCCGTGCTC 5´

Lambda ZAP® II Premade Library 5

BACTERIAL HOST STRAINS

Host Strain Genotypes

Host strain Genotype

SOLR™ straina e14–(McrA–) Δ(mcrCB-hsdSMR-mrr)171 sbcC recB recJ uvrC

umuC::Tn5 (Kanr) lac gyrA96 relA1 thi-1 endA1 λR [F´ proAB lacI

XL1-Blue MRF´ strain Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1

recA1 gyrA96 relA1 lac [F´ proAB lacI

Use the SOLR strain for excision only.

XL1-Blue MRF’ Bacterial Strain Description

The RecA– E. coli host strain XL1-Blue MRF´ is supplied with the Lambda ZAP II library. Because the Lambda ZAP II vector does not require a supF genotype, the amplified library grows very efficiently on the XL1-Blue MRF´ strain. important when working with the Lambda ZAP II vector as the F´ episome present in the XL1-Blue MRF´ strain 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 II 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 Lambda ZAP II Vector).

ZΔM15] Su– (nonsuppressing)

ZΔM15 Tn10 (Tetr)]

In addition, use of the correct host strain is

Phagemid from the

6 Lambda ZAP® II 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, XL1-Blue MRF´ 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

repressor in the E. coli host strain

and Y1090) are not suitable for use with the Lambda ZAP II 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 II vector. Using these strains with the Lambda ZAP II vector could result in recombination between the homologous sequences.

Host strain

SOLR™ strain LB-kanamycina LB broth with supplements

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

See Preparation of Media and Reagents.

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

Maltose and magnesium supplements are required for optimal lambda phage receptor expression on the surface of the XL1-Blue MRF’ 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

Agar plates and top agar for plaque formation

— LB-ampicillina

NZYa —

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.

Lambda ZAP® II Premade Library 7

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.

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.)

. Before use, dilute cells to an OD

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

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 II 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 II vector, is partly responsible for the reduced activity of the

β-galactosidase protein. As would be expected, the copy number of the Lambda ZAP II 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.

8 Lambda ZAP® II Premade Library

HELPER PHAGE

Two different helper phages are provided with the Lambda ZAP II library: (1) the ExAssist and (2) the VCSM13 helper phage. The ExAssist interference-resistant helper phage with SOLR strain is designed to allow efficient in vivo excision of the pBluescript phagemid from the Lambda ZAP II vector while preventing the problems that can be associated with helper phage coinfection. The ExAssist helper phage contains an amber mutation that prevents replication of the phage genome in a nonsuppressing E. coli strain (e.g., SOLR cells). Only the excised phagemid can replicate in the host, removing the possibility of co-infection from the ExAssist helper phage. The ExAssist helper phage cannot be used for single-stranded rescue due to its inability to replicate in the SOLR strain. The other helper phage, VCSM13 helper phage, is recommended for single-stranded rescue procedures from the excised pBluescript phagemids.

Storing the Helper Phage

The ExAssist 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 the VCSM13 helper phage. If the titer drops over time, prepare a fresh hightiter stock of the helper phage as outlined in Amplifying the Helper Phage.

interference-resistant helper phage with SOLR™ strain3

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

Titering 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

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.

of 1.0.

600

–4

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

= 1.0).

600

Lambda ZAP® II Premade Library 9

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

(

Note ExAssist and VCSM13 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.

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

)

Volume plated l

(

⎤

1000 l / ml

)

⎥ ⎦

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

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.

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

1.0 × 10

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

pfu/ml for VCSM13 helper phage.

and

Note ExAssist and VCSM13 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

Titering the Helper Phage or Reference 4.

see

10 Lambda ZAP® II Premade Library

TITERING THE LIBRARY

Preparing the Host Bacteria

1. Streak the XL1-Blue MRF’ cells onto an LB-tetracycline agar plate.

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.

Incubate the plate overnight at 37°C.

single colony of the XL1-Blue MRF’ 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.

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.

Titering Protocol

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

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

10 mM MgSO

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. Stratagene suggests adding the IPTG and X-gal to the NZY top agar separately, with mixing in between additions, to minimize the formation of this precipitate. Plate immediately on NZY agar plates. Plaques are visible after incubation for 12 hours at 37°C, although color detection requires overnight incubation. Background plaques are blue, while recombinant plaques are white.

1. Dilute the XL1-Blue MRF’ cells (from step 5 of

Bacteria

10 mM MgSO

in Titering the Library) to an OD

Preparing the Host

of 0.5 with sterile

600

Note The bacteria should be used immediately following dilution.

Lambda ZAP® II Premade Library 11

2. To determine the titer of the library, mix the following components:

1 μl of the library aliquot 200 μl of XL1-Blue MRF’ cells at an OD

of 0.5

600

and

1 μl of a 1:10 dilution of the library aliquot 200 μl of XL1-Blue MRF’ cells at an OD

of 0.5

600

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.

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 12 hours, although color detection requires overnight incubation. Background plaques are blue and should

be < 1× 10

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

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

12 Lambda ZAP® II Premade Library

AMPLIFYING THE LIBRARY

It is usually desirable to amplify libraries prepared in lambda vectors to make a large, stable quantity of a high-titer stock of the library. However, more than one round of amplification is not recommended, since slower growing clones may be significantly underrepresented.

Note The premade library has been through one round of amplification.

The following protocol is recommended for amplifying the Lambda ZAP II library:

Day 1

1. Grow a 50-ml overnight culture of XL1-Blue MRF’ cells in LB broth with supplements at 30°C with shaking.

Day 2

2. Gently spin down the XL1-Blue MRF’ cells (1000 × g). Resuspend the cell pellet in 25 ml of 10 mM MgSO suspension, then dilute the cells to an OD

. Measure the OD

of 0.5 in 10 mM MgSO4.

600

of the cell

600

3. Combine aliquots of the library suspension containing ~5 × 10 bacteriophage with 600 μl of XL1-Blue MRF’ cells at an OD

in Falcon a total of 20 aliquots (each aliquot contains 5 × 10

2059 polypropylene tubes. To amplify 1 × 106 plaques, use

plaques/150-mm

pfu of

of 0.5

600

plate).

Note Do not add more than 300 μl of phage/600 μl of cells.

4. Incubate the tubes containing the phage and host cells for 15 minutes at 37°C to allow the phage to attach to the cells.

5. Mix 6.5 ml of NZY top agar, melted and cooled to ~48°C, with each aliquot of infected bacteria and spread evenly onto a freshly poured 150-mm NZY agar plate. Allow the plates to set for 10 minutes.

6. Invert the plates and incubate at 37°C for 6–8 hours. Do not allow the plaques to get larger than 1–2 mm. On completion, the plaques should be touching.

7. Overlay the plates with ~8–10 ml of SM buffer. Store the plates at 4°C overnight (with

gentle rocking if possible). This allows the phage to

diffuse into the SM buffer.

Lambda ZAP® II Premade Library 13

Day 3

8. Recover the bacteriophage suspension from each plate and pool it into a sterile polypropylene container. Rinse the plates with an additional 2 ml of SM buffer and pool. Add chloroform to a 5% (v/v) final concentration. Mix well and incubate for 15 minutes at room temperature.

9. Remove the cell debris by centrifugation for 10 minutes at 500 ×

10. Recover the supernatant and transfer it to a sterile polypropylene container. If the supernatant appears cloudy or has a high amount of cell debris, repeat steps 8 and 9. If the supernatant is clear, add chloroform to a 0.3% (v/v) final concentration and store at 4°C. Stratagene recommends storing aliquots of the amplified library in 7% (v/v) DMSO at –80°C.

11.

Check the titer of the amplified library using host cells and serial

dilutions of the library. (Assume ~10

–1011 pfu/ml.)

Note Briefly spin the lambda phage stock to ensure that the

chloroform is separated completely before removing the aliquot for titering.

14 Lambda ZAP® II Premade Library

PERFORMING PLAQUE LIFTS

1. Titer the library suspension to determine the concentration using XL1-Blue MRF’ cells.

2. Combine the equivalent of 5 × 10 prepared XL1-Blue MRF’ cells at an OD

pfu/plate and 600 μl of freshly

of 0.5.

600

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

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® II Premade Library 15

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

10. Crosslink the DNA to the membranes using the autocrosslink setting on the Stratalinker

~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

et al.

for suggested phage miniprep and maxiprep procedures.

ANTIBODY SCREENING PROTOCOL

A complete manual for immunoscreening is supplied with Stratagene's

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

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

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

UV crosslinker* (120,000 μJ of UV energy) for

4, 5

Following

4, 5

After an isolate is obtained, refer to

16 Lambda ZAP® II Premade Library

In Vivo EXCISION OF THE pBLUESCRIPT LAMBDA ZAP II VECTOR

The Lambda ZAP II 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 (see Figure

3). 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. ZAP II vector. The lambda phage (target) is made accessible to the f1-derived proteins by simultaneously infecting a strain of the lambda vector and 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´ of 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 II 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.

These two regions are subcloned separately into the Lambda

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

SK(–) PHAGEMID FROM THE

E. coli with both

Lambda ZAP® II Premade Library 17

In Vivo EXCISION PROTOCOLS USING THE EXASSIST R

ESISTANT HELPER PHAGE WITH SOLR™ STRAIN

The ExAssist helper phage with SOLR strain is designed to allow efficient excision of the pBluescript phagemid from the Lambda ZAP II vector, while eliminating problems associated with helper phage co-infection. The ExAssist helper phage contains an amber mutation that prevents replication of the phage genome in a nonsuppressing This allows only the excised phagemid to replicate in the host, removing the possibility of co-infection from the ExAssist helper phage. Since the ExAssist helper phage cannot replicate in the SOLR strain, single-stranded rescue cannot be performed in this strain using this helper phage.

Mass excision can be used to generate subtraction libraries and subtracted DNA probes.

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.)

E. coli strain such as SOLR cells.

INTERFERENCE-

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

Day 2

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

of the cell suspensions, then adjust the concentration of the

600

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

600

4. Combine the following components in a Falcon 2059 polypropylene tube:

200 μl of XL1-Blue MRF´ cells at an OD 250 μl of phage stock (containing >1 × 10 1 μl of the ExAssist helper phage (>1 × 10

of 1.0

600

phage particles)

pfu/μl)

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 Falcon 2059 polypropylene tube at 37°C for 15 minutes to allow the phage to attach to the cells.

. Measure

18 Lambda ZAP® II Premade Library

6. Add 3 ml of LB broth with supplements and incubate the Falcon 2059 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 Falcon 2059 polypropylene tube at 65–70°C for 20 minutes to lyse the lambda phage particles and the cells. Spin the tube at 1000 ×

for 15 minutes to pellet the cell debris.

8. Decant the supernatant into a sterile Falcon 2059 polypropylene 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.)

9. To plate the excised phagemids, add 200 μl of freshly grown SOLR cells from step 3 (OD

= 1.0) to two 1.5-ml microcentrifuge tubes.

600

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 titrate the supernatant to achieve single-colony isolation.

Colonies appearing on the plate contain the pBluescript double-stranded phagemid with the cloned DNA insert. Helper phage will not grow, since helper phage is unable to replicate in the Su

–

(nonsuppressing) SOLR strain and does not contain ampicillin-resistance genes. SOLR cells are also resistant to lambda phage infection, thus preventing lambda phage contamination after excision.

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

Recovery Of Single-Stranded DNA From Cells Containing pBluescript Phagemids

Appendix:

Lambda ZAP® II Premade Library 19

Mass Excision Protocol

Day 1

1. Grow separate 50-ml overnight cultures of XL1-Blue MRF´ and

SOLR cells in LB broth with supplements at 30°C.

Day 2

2. Gently spin down the XL1-Blue MRF´ and SOLR 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 XL1-Blue MRF´ cells at a MOI of 1:10 lambda phage-to-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 ExAssist 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

. Measure

of the cell suspensions, then adjust the concentration of the

600

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

600

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

XL1-Blue MRF´ cells (1:10 lambda phage-to-cell ratio, noting that an OD

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

of 1.0 corresponds to 8 × 108 cells/ml)

600

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.

20 Lambda ZAP® II Premade Library

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

200 μl of SOLR 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® II Premade Library 21

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

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,

the F´ episome.

Stratagene offers helper phages that 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 ~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 in vitro without further subcloning.

VCSM13 (single-strand size ~6 kb), is efficient at single-stranded DNA rescue and provides good yields of single-stranded phagemid; however it can revert to wild-type (more frequently than R408 helper phage, for example). 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.]

PHAGEMIDS

phenotype (containing an F´ episome), pBluescript

E. coli without an F´ episome coding for

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

7, 8

preferentially package pBluescript

22 Lambda ZAP® II 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 helper phage at 10 (MOI ~10).

2. Grow the culture at 37°C with vigorous aeration for 1–2 hours.

3. Add kanamycin to 70 μg/ml to select for infected cells.

4. Continue growth at 37°C with vigorous aeration for 16–24 hours, or

until growth has reached saturation.

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

6. 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.

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

be obvious.)

–108 pfu/ml

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

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

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

1 mM EDTA by vortexing vigorously.

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

1–2 minutes to separate phases.

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

Centrifuge for 5 minutes.

12. Remove ethanol and dry the DNA pellet.

13. Dissolve the pellet in 25 μl of TE buffer

14.

Analyze 1–2 μl on an agarose gel.

See Preparation of Media and Reagents.

Lambda ZAP® II Premade Library 23

TROUBLESHOOTING

Observation Suggestions

The number of colonies is too low

* ABLE competent cells (Catalog #200170–200172) and ABLE electroporation competent cells (Catalog #200160–

200162) are available separately from Stratagene.

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

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

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

24 Lambda ZAP® II Premade Library

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

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 1

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

LB–Tetracycline Broth (per Liter)

Prepare 1 liter of LB broth Autoclave Cool to 55°C Add 1.5 ml of 10 mg/ml tetracycline (filter-

sterilized) 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 ampicillin

(filter-sterilized)

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

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

LB–Tetracycline Agar (per Liter)

Prepare 1 liter of LB agar Autoclave Cool to 55°C Add 1.5 ml of 10 mg/ml tetracycline (filter-

sterilized) 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

LB–Kanamycin Broth (per Liter)

Prepare 1 liter of LB broth Autoclave Cool to 55°C Add 7.5 ml of 10 mg/ml kanamycin (filter-

sterilized)

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)

Lambda ZAP® II Premade Library 25

2× YT Broth (per Liter)

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

liter Adjust to pH 7.5 with NaOH Autoclave

O to a final volume of 1

NZY Broth (per Liter)

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

liter Adjust the pH to 7.5 with NaOH Autoclave

. 7H2O

O to a final volume of 1

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)

. 7H2O

O to a final volume of

TE Buffer

5 mM Tris-HCl (pH 7.5)

0.1 mM EDTA

SM Buffer (per Liter)

5.8 g of NaCl

2.0 g of MgSO

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

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

liter

Autoclave

· 7H2O

O to a final volume of 1

NZY Top Agar (per Liter)

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

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

1 liter

26 Lambda ZAP® II Premade Library

REFERENCES

ENDNOTES

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

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

3. Hay, B. and Short, J. M. (1992) Strategies 5(1):16–18.

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.

ABLE®, ExAssist®, Lambda ZAP®, pBluescript®, and Stratalinker® are registered

trademarks of Stratagene in the United States.

picoBlue and SOLR are trademarks of Stratagene.

Falcon

is a registered trademark of Becton Dickinson Labware. GenBank Parafilm Pyrex Whatman

is a registered trademark of the U.S. Department of Health and Human Services.

is a registered trademark of American Can Company.

is a registered trademark of Corning Glass Works.

is a trademark of Whatman Paper Ltd.

MSDS INFORMATION

The Material Safety Data Sheet (MSDS) information for Stratagene products is provided on Stratagene’s website at MSDS’s in a print-ready format. MSDS documents are not included with product shipments.

http://www.stratagene.com/MSDS/. Simply enter the catalog number to retrieve any associated

Lambda ZAP® II Premade Library 27

Sutter Instrument ZAP II Instruction Manual

Specifications and Main Features

Frequently Asked Questions

User Manual