Sutter Instrument EMBL3 User Manual

Lambda EMBL3/BamH I
Vector Kit

INSTRUCTION MANUAL

Catalog #241211 (Lambda EMBL3/BamH I Vector Kit) and

#241612 (Lambda EMBL3/BamH I Gigapack III Gold Cloning Kit)

Revision A

For In Vitro Use Only

241211-12

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Lambda EMBL3/BamH I Vector Kit

CONTENTS

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

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

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

Overview of the Lambda EMBL3 Vector System ................................................................ 2

Lambda EMBL3 Vector Map................................................................................................ 2

Preparing the Host Strains................................................................................................................. 3

Host Strain Genotypes...........................................................................................................3

Growing and Maintaining the Host Strains ........................................................................... 3

Preparing a –80°C Bacterial Glycerol Stock ......................................................................... 4

Ligating the Insert............................................................................................................................... 5

Packaging............................................................................................................................................. 6

General Information ..............................................................................................................6

Gigapack III XL Packaging Extract ......................................................................................6

Packaging Instructions........................................................................................................... 7

Titering Procedure .............................................................................................................................. 9

Amplifying the Library..................................................................................................................... 10

Day 1 ...................................................................................................................................10

Day 2 ...................................................................................................................................10

Day 3 ...................................................................................................................................11

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

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

Troubleshooting ................................................................................................................................ 13

Preparation of Media and Reagents................................................................................................ 14

References .......................................................................................................................................... 15

Endnotes............................................................................................................................................. 15

MSDS Information............................................................................................................................ 15

Lambda EMBL3/BamH I Vector Kit

ATERIALS PROVIDED

M

Quantity

Materials provided

Lambda EMBL3 vector double-digested with EcoR I and BamH I,
CIAP-treated

pME/BamH I test insert (~12 kb)b 2.5 μg 2.5 μg

Host strains

XL1-Blue MRA 0.5-ml bacterial

XL1-Blue MRA (P2) 0.5-ml bacterial

Gigapack III Gold-11 packaging extracte — 11 × 25 μl

λcI857 Sam7 wild-type lambda control DNAf — 1.05 μg

VCS257 host straing — 1 ml

a

Shipped as a liquid at 1 μg/μl in 5 mM Tris-HCl (pH 7.5) and 0.1 mM EDTA. On arrival, store the EMBL3 vector at

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

b

Shipped as a liquid at 0.25 μg/μl in 5 mM Tris-HCl (pH 7.5) and 0.1 mM EDTA. On arrival, store the pME/BamH I test

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

c

For host strain shipping and storage conditions, please see Preparing the Host Strains.

d

The XL1-Blue MRA and XL1-Blue MRA (P2) strains have been modified to enhance the stability of clones containing

methylated DNA, as well as nonstandard DNA structures.

e

Gigapack III packaging extract is very sensitive to slight variations in temperature. Storing the packaging extracts at the

bottom of a –80°C freezer directly from the dry ice shipping container is required in order to prevent a loss of packaging
efficiency. Transferring tubes from one freezer to another may also result in a loss of efficiency. Do not allow the
packaging extracts to thaw! Do not store the packaging extracts in liquid nitrogen as the tubes may explode.

f

The λcI857 Sam7 wild-type lambda control DNA is shipped frozen and should be stored at –80°C immediately on receipt.

g

The VCS257 host strain, included for plating the λcI857 Sam7 positive control, is shipped as a frozen bacterial glycerol
stock (see Preparing the Host Strains for additional storage instructions) and should also be stored at –80°C immediately
on receipt. This control host strain is a derivative of DP50 supF and should be used only when plating the packaged test
DNA. The control DNA used with Gigapack III Gold packaging extract requires a supF mutation in the bacterial host to
plate efficiently.

a

c,d

Catalog #241211 Catalog #241612

10 μg 10 μg

0.5-ml bacterial

glycerol stock

glycerol stock

glycerol stock

0.5-ml bacterial
glycerol stock

STORAGE CONDITIONS

Lambda EMBL3 Vector: –20°C
Test Insert: –20°C
Bacterial Glycerol Stocks: –80°C
Packaging Extracts: –80°C

Revision A © Agilent Technologies, Inc. 2008.

Lambda EMBL3/BamH I Vector Kit 1

INTRODUCTION

Overview of the Lambda EMBL3 Vector System

The Lambda EMBL3 vector is a genomic replacement lambda phage vector1
capable of accepting BamH I-compatible fragments (Sau3A I, Mbo I, Bgl II,
or BamH I) ranging in size from 9 to 23 kb (Figure 1). The arms are
prepared by double digestion with BamH I and EcoR I followed by a
selective precipitation which removes the small BamH I/EcoR I linker that
separates the arms from the stuffer fragment. Because this treatment leaves
the arms with BamH I ends and the stuffer fragment with EcoR I ends, there
is no need to physically separate them. Target DNA cloned into the
BamH I sites of EMBL3 may be removed by digestion with Sal I.

The Lambda EMBL3 system takes advantage of spi (sensitive to
P2 inhibition) selection. Lambda phages containing active red and gam
genes are unable to grow on host strains that contain P2 phage lysogens.
Lambda phages without these genes are able to grow on strains lysogenic
for P2 such as XL1-Blue MRA (P2), a P2 lysogen of XL1-Blue MRA. The
red and gam genes in Lambda EMBL3 DNA are located on the stuffer
fragment; therefore, wild-type Lambda EMBL3 phage cannot grow on
XL1-Blue MRA (P2). When the stuffer fragment is replaced by an insert,
the recombinant Lambda EMBL3 becomes red
to grow on the P2 lysogenic strain. Therefore, by plating the library on the
XL1-Blue MRA (P2) strain, only recombinant phages are allowed to grow.
The strain XL1-Blue MRA is also provided as a control strain and later for
growth of the recombinant when the selection is no longer necessary.

–

/gam–, and the phage is able

Lambda EMBL3 Vector Map

FIGURE 1 Map of the Lambda EMBL3 replacement vector.

2 Lambda EMBL3/BamH I Vector Kit

PREPARING THE HOST STRAINS

Host Strain Genotypes

Host strain Genotype

XL1-Blue MRA strain Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 gyrA96 relA1 lac

XL1-Blue MRA (P2) strain XL1-Blue MRA (P2 lysogen)

Growing and Maintaining the Host Strains

The bacterial host strains are shipped as bacterial glycerol stocks. For the
appropriate media, please refer to the following table:

Host strain

XL1-Blue MRA strain LB LB LB with 0.2% (w/v) maltose–10 mM MgSO4

XL1-Blue MRA (P2) strain LB LB LB with 0.2% (w/v) maltose–10 mM MgSO4

VCS257 strainb LB LB LB with 0.2% (w/v) maltose–10 mM MgSO4

a

The XL1-Blue MRA and XL1-Blue MRA (P2) host strains are modified to enhance the stability of clones containing

methylated DNA; in addition, these strains enhance the stability of nonstandard DNA structures.

b

For use with Gigapack III packaging extract and wild-type control only. Supplied with Gigapack III packaging extract.

Agar plates for

bacterial streak

Medium for

bacterial

§

glycerol stock§

Medium for bacterial cultures for

titering phage (final concentration)

On arrival, prepare the following from the bacterial glycerol stock using the
appropriate media as indicated in the previous table:

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 also 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 (see Preparation of Media
and Reagents).

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.

§

See Preparation of Media and Reagents.

Lambda EMBL3/BamH I Vector Kit 3

Preparing a –80°C Bacterial Glycerol Stock

1. In a sterile 50-ml conical tube, inoculate 10 ml of appropriate liquid
medium 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 (5 ml of
glycerol + 5 ml of 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.

4 Lambda EMBL3/BamH I Vector Kit

LIGATING THE INSERT

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

Prepare a ligation reaction mixture containing the following components:

1.0 μl of predigested Lambda EMBL3/BamH I (1 μg)

1.2 μl of pME/BamH I insert (0.3 μg)

0.5 μl of 10× ligase buffer

0.5 μl of 10 mM rATP (pH 7.5)

2 U of T4 DNA ligase
Water up to a final volume of 5 μl

Incubate the ligation at 4°C overnight.

When ligating your own insert, you may use up to 2.5 μl in volume. Use an
equal molar ratio of your BamH I-compatible insert DNA (Sau3A I, Mbo I,
Bgl II, or BamH I) with the Lambda EMBL3 arms. The Lambda EMBL3
vector can accommodate inserts ranging from 9 to 23 kb. If ligating a

20,000-bp insert to the arms, use 0.4 μg of insert for every 1 μg of arms. If
the insert used is free from contaminants and contains a high percentage of
ligatable ends, expect about 1 × 10
using high-efficiency packaging extract, such as Gigapack III Plus or
Gigapack III Gold packaging extracts.*

5% (v/v). Do not exceed 5% (v/v) glycerol. Due to the high
molecular weight of the lambda vector, the contents may be very
viscous. It is important to microcentrifuge the contents of the
lambda vector tube briefly at 11,000 × g and then to mix the
solution gently by stirring with a yellow pipet tip prior to
pipetting.

§

6

–1.5 × 107 recombinant plaques when

Note The Lambda EMBL3 vector arms provided have been pre-treated

with calf intestine alkaline phosphatase

(CIAP). Do not CIAP-treat
the insert DNA. We recommend size fractionation of the insert
DNA to minimize cloning of multiple inserts.

§

See Preparation of Media and Reagents.

*Gigapack III Gold packaging extract [Catalog #200201 (Gold-4), #200202 (Gold-7), and

#200203 (Gold-11)]. Gigapack III Plus packaging extract [Catalog #200204 (Plus-4),
#200205 (Plus-7), and #200206 (Plus-11)]

Lambda EMBL3/BamH I Vector Kit 5

PACKAGING

General Information

Packaging extracts are used to package recombinant lambda phage with
high efficiency. The single-tube format of Gigapack III packaging extract
simplifies the packaging procedure and increases the efficiency and
representation of libraries constructed from highly methylated DNA. Each
packaging extract is restriction minus (HsdR

–

McrA– McrBC– McrF– Mrr–)
to optimize packaging efficiency and library representation. When used in
conjunction with restriction-deficient plating cultures, Gigapack III
packaging extract improves the quality of DNA libraries constructed from
methylated DNA.

2–5

Optimal packaging efficiencies are obtained with lambda DNAs that are
concatemeric. Ligations should be carried out at DNA concentrations of

0.2 μg/μl or greater, which favors concatemers and not circular DNA
molecules that only contain one cos site. DNA to be packaged should be
relatively free from contaminants. Polyethylene glycol (PEG), which is
contained in some ligase buffers, can inhibit packaging. The volume of

DNA added to each extract should be between 1 and 4 μl. To obtain the
highest packaging efficiency [i.e., the number of plaque-forming units per

microgram (pfu/μg) of DNA], package 1 μl of the ligation reaction and
never more than 4 μl. Increased volume (i.e., >4 μl) yields more plaque-

forming units per packaging reaction, but fewer plaque-forming units per
microgram of DNA.

DNA that is digested with restriction enzymes and religated packages less
efficiently (by a factor of 10–100) than uncut lambda DNA. For example,
uncut wild-type lambda DNA packages with efficiencies exceeding

1 × 10

9

pfu/μg of vector when using a Gigapack III packaging extract.

However, predigested vector, when ligated to a test insert, yields
~5 × 10

6

– 1 × 107 recombinant plaques/μg of vector.

Gigapack III XL Packaging Extract

Gigapack III XL packaging extract (Gigapack III XL packaging extract
[Catalog #200207 (XL-4), #200208 (XL-7), and #200209 (XL-11)]) is an
in vitro packaging extract, which preferentially size selects for extra large
inserts, while maintaining the highest packaging efficiencies commercially
available. This extract is specifically designed for use in generating genomic
libraries. For example, a 20-kb insert will be packaged with a 95% higher
efficiency than a 14-kb insert when using replacement vectors such as the
Lambda EMBL3 vector.

6 Lambda EMBL3/BamH I Vector Kit

Packaging Instructions

For optimal packaging efficiency, package 1 μl of the ligation and never
more than 4 μl. For further selection of large inserts, we recommend using

Gigapack III XL packaging extract, a size-selective packaging extract.

Preparing the Host Bacteria

Note Prepare an overnight culture of the VCS257 strain (see the table

1. Streak the bacterial glycerol stock onto the appropriate agar plates (see

the table in Preparing the Host Strains). Incubate the plates overnight
at 37°C.

in Preparing the Host Strains) prior to performing the protocol for
the positive wild-type lambda DNA control (see Positive Wild-

Type Lambda DNA Control for the Gigapack III Packaging
Extract).

2. Inoculate an appropriate medium, supplemented with 10 mM MgSO

4

and 0.2% (w/v) maltose, with a single colony.

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

of 1.0). Alternatively, grow overnight at 30°C, shaking at 200

OD

600

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.

4. Pellet the bacteria at 500 × g for 10 minutes.

5. Gently resuspend the cells in half the original volume with sterile

10 mM MgSO

6. Dilute the cells to an OD

.

4

of 0.5 with sterile 10 mM MgSO4.

600

Note The bacteria should be used immediately following dilution.

Packaging Protocol

Note Polyethylene glycol, which is contained in some ligase buffers, can

inhibit packaging.

1. Remove the appropriate number of packaging extracts from a

–80°C freezer and place the extracts on dry ice.

2. Quickly thaw the packaging extract by holding the tube between your

fingers until the contents of the tube just begins to thaw.

3. Add the experimental DNA immediately (1–4 μl containing

0.1–1.0 μg of ligated DNA) to the packaging extract.

Lambda EMBL3/BamH I Vector Kit 7

4. Stir the tube with a pipet tip to mix well. Gentle pipetting is allowable

provided that air bubbles are not introduced.

5. Spin the tube quickly (for 3–5 seconds), if desired, to ensure that all

contents are at the bottom of the tube.

6. Incubate the tube at room temperature (22°C) for 2 hours. Do not

exceed 2 hours.

Note The highest efficiency occurs between 90 minutes and

2 hours. Efficiency may drop dramatically during extended
packaging times.

§

7. Add 500 μl of SM

buffer to the tube.

8. Add 20 μl of chloroform and mix the contents of the tube gently. Spin

the tube briefly to sediment the debris.

9. The supernatant containing the phage is ready for titering. The

supernatant may be stored at 4°C for up to 1 month.

Testing the Efficiency of Gigapack III Packaging Extract
with the Wild-Type Lambda Control DNA (Optional)

Use the following procedure to test the efficiency of the Gigapack III
packaging extract with the λcI857 Sam7 wild-type lambda control DNA:

1. Thaw the frozen wild-type lambda control DNA on ice and gently mix

after thawing.

2. Using 1 μl of the wild-type lambda control DNA (~0.2 μg), proceed

with steps 1–10 in the Packaging Protocol.

Note Because of the high titer achieved with the wild-type lambda

control DNA, Stop the control packaging reaction with 1 ml
of SM buffer. This should make the plaques easier to count.

–2

3. Prepare two consecutive 10

dilutions of the packaging reaction from

step 10 in the Packaging Protocol in SM buffer. (The final dilution is

–4

.)

10

4. Add 10 μl of the 10

–4

dilution to 200 μl of the VCS257 host strain.

(This strain is recommended for plating the wild-type lambda control
DNA only.) Incubate at 37°C for 15 minutes. Add 3 ml of NZY top

§

, melted and cooled to ~48°C, and quickly pour the dilution onto

agar
dry, prewarmed NZY agar plates.

5. Incubate the plates for at least 12 hours at 37°C. Count the plaques.

–4

Approximately 400 plaques should be obtained on the 10

dilution

plate when the reaction is stopped with 1 ml of SM buffer.

§

See Preparation of Media and Reagents.

8 Lambda EMBL3/BamH I Vector Kit

TITERING PROCEDURE

1. Streak the bacterial glycerol stock onto the appropriate agar plates (see

the table in Preparing the Host Strains). Incubate the plates overnight
at 37°C.

2. Inoculate an appropriate medium, supplemented with 10 mM MgSO

4

and 0.2% (w/v) maltose, with a single colony.

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

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

OD

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.

4. Pellet the bacteria at 500 × g for 10 minutes.

5. Gently resuspend the cells in half the original volume with sterile

10 mM MgSO

6. Dilute the cells to an OD

.

4

of 0.5 with sterile 10 mM MgSO4.

600

Note The bacteria should be used immediately following dilution.

7. Prepare dilutions of the final packaged reaction in SM buffer. Add

1 μl of the final packaged reaction to 200 μl of host cells diluted in
10 mM MgSO

to an OD

4

of 0.5. If desired, also add 1 μl of a

600

1:10 dilution of the packaged reaction in SM buffer to 200 μl of host
cells.

8. Incubate the phage and the bacteria at 37°C for 15 minutes to allow the

phage to attach to the cells.

9. Add 3 ml of NZY top agar, melted and cooled to ~48°C, and plate

immediately on prewarmed NZY agar plates.

Lambda EMBL3/BamH I Vector Kit 9

10. Count the plaques and determine the titer in plaque-forming units per

milliliter (pfu/ml).

Both recombinant and nonrecombinant phage will grow on XL1-Blue MRA,
but only recombinant phage will grow on XL1-Blue MRA (P2). Plaques
should be visible after 8–12 hours of incubation at 37°C.

Note Historically, the host strain LE392 has been used with this vector;

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.

however, E. coli restriction systems in this strain have a
significant negative effect on the efficiency of DNA cloning and the
ability to generate libraries representative of the gene population.
The strains provided, XL1-Blue MRA and XL1-Blue MRA (P2),
are mcrA

–

, mcrB– and mrr–; these modifications have been
demonstrated to cause up to a 10-fold increase in the yield of
recombinant phage containing methylated DNA. In addition, these
strains have been further modified to enhance the stability of
nonstandard DNA structures. Due to the removal of the red/gam
genes during the preparation of the replacement vector phage with
insert DNA is unable to plate on a recA

–

host strain.

Day 1

Day 2

1. Prepare the host strains as outlined in Preparing the Host Strains.

2. Dilute the cells to an OD
cells at an OD

of 0.5/150-mm plate.

600

of 0.5 in 10 mM MgSO4. Use 600 μl of

600

3. Combine aliquots of the packaged mixture or library suspension
containing ~5 × 10

of 0.5 in 14-ml BD Falcon polypropylene round-bottom tubes

OD

600

(BD Biosciences Catalog #352059). To amplify 1 × 10
total of 20 aliquots (each aliquot contains 5 × 10

4

pfu of bacteriophage with 600 μl of host cells at an

6

plaques, use a

4

plaques/150-mm

plate).

μ

Note Do not add more than 300

l of phage/600 μl of cells.

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

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 bottom agar plate.

10 Lambda EMBL3/BamH I Vector Kit

Day 3

6. Incubate the plates 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.

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 × g.

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. Store
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

PERFORMING PLAQUE LIFTS

1. Titer the library to determine the concentration (prepare fresh host cells
to use in titering and in screening).

2. Plate on large 150-mm NZY agar plates (≥2-day-old) to 50,000
pfu/plate with 600 μl of host cells at an OD

NZY top agar/plate. (Use 20 plates to screen 1 × 10

3. Incubate the plates at 37°C for ~8 hours.

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

9

–1011 pfu/ml.)

of 0.5/plate and 6.5 ml of

600

6

.)

Note Use forceps and wear gloves for the following steps.

5. Transfer the plaques onto a nitrocellulose membrane (Stratagene
Catalog #420106–#420108) for 2 minutes. Use a needle to prick
through the agar for orientation. (If desired, waterproof ink in a syringe
needle may be used.)

If making duplicate nitrocellulose membranes, allow the second

membrane to transfer for ~4 minutes.

Lambda EMBL3/BamH I Vector Kit 11

Note Pyrex® dishes are convenient for the following steps. All

solutions should be at room temperature.

a. Denature the nitrocellulose membrane 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.

§

6. Blot briefly on a Whatman

7. 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.

8. 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.

§

See Preparation of Media and Reagents.

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

7

®

3MM paper.

6,7

Following

6,7

After an isolate is obtained, refer to

for suggested phage miniprep and maxiprep procedures.

12 Lambda EMBL3/BamH I Vector Kit

TROUBLESHOOTING

Observations Suggestions

Packaging efficiency is too low

During titering, neither a bacterial
lawn nor plaques is observed on the
plate

Ensure that the packaging extracts are properly stored. Gigapack III packaging
extract is very sensitive to slight variations in temperature; therefore, store the
packaging extracts at the bottom of a –80°C freezer and avoid transferring tubes
from one freezer to another. Do not allow the packaging extracts to thaw

Avoid the use of ligase buffers containing PEG, which can inhibit packaging

The DNA concentration in the packaging extract may be too low. Ligate at DNA
concentrations of 0.2 μg/μl or greater and package between 1 and 4 μl of the
ligation reaction

Packaging extract protein concentration may be too low. Never package >4 μl of
the ligation reaction to prevent dilution of the proteins contained within the
packaging extract

Chloroform, added after packaging to prevent bacterial contamination, may be
present while titering. Be sure to spin down the chloroform completely prior to
removing an aliquot of the viral stock for titering

Lambda EMBL3/BamH I Vector Kit 13

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)

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

1 liter
Adjust the pH to 7.5 with NaOH
Autoclave

. 7H2O

4

O to a final volume of

2

LB Broth (per Liter)

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

O to a final volume of

2

1 liter

Adjust to pH 7.0 with 5 N NaOH
Autoclave

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

4

O to a final volume of

2

10× Ligase Buffer

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

2

10 mM dithiothreitol (DTT)

Note rATP is added separately in
the ligation reaction

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

1 liter

Autoclave

· 7H2O

4

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

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

14 Lambda EMBL3/BamH I Vector Kit

REFERENCES

ENDNOTES

1. Frischauf, A. M., Lehrach, H., Poustka, A. and Murray, N. (1983) J Mol Biol
170(4):827-42.

2. Kohler, S. W., Provost, G. S., Kretz, P. L., Dycaico, M. J., Sorge, J. A. et al. (1990)
Nucleic Acids Res 18(10):3007-13.

3. Kretz, P. L., Kohler, S. W. and Short, J. M. (1991) J Bacteriol 173(15):4707-16.

4. Kretz, P. L., Reid, C. H., Greener, A. and Short, J. M. (1989) Nucleic Acids Res
17(13):5409.

5. Kretz, P. L. and Short, J. M. (1989) Strategies in Molecular Biology 2(2):25-26.

6. 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.

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

Parafilm® is a registered trademark of American Can Company.

®

Pyrex

is a registered trademark of Corning Glass Works.

Whatman

®

is a registered trademark of Whatman Ltd.

MSDS INFORMATION

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

Lambda EMBL3/BamH I Vector Kit 15