Gigapack Gigapack III Gold, Gigapack III Plus, Gigapack III XL User Guide

Gigapack III Gold Packaging Extract, Gigapack III
Plus Packaging Extract, and Gigapack III XL
Packaging Extract

Instruction Manual

Catalog #200201 (Gigapack III Gold-4), #200202 (Gigapack III Gold-7),

and #200203 (Gigapack III Gold-11)

Catalog #200204 (Gigapack III Plus-4), #200205 (Gigapack III Plus-7),

and #200206 (Gigapack III Plus-11)

Catalog #200207 (Gigapack III XL-4), #200208 (Gigapack III XL-7),

and #200209 (Gigapack III XL-11)

Revision B

Research Use Only. Not for Use in Diagnostic Procedures.

200201-12

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 Agilent. Agilent 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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Gigapack III Gold Packaging Extract, Gigapack III Plus
Packaging Extract, and Gigapack III XL Packaging Extract

CONTENTS

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

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

Introduction ......................................................................................................................................... 2

Preparation of Host Strains ................................................................................................................ 3

Preparation of a –80°C Bacterial Glycerol Stock .................................................................. 3

Preparation of Host Bacteria ................................................................................................. 4

Packaging Protocol ............................................................................................................................. 4

Positive Wild-Type Lambda DNA Control for the Gigapack III Packaging Extract ............ 5

Titering the Packaging Reaction ........................................................................................................ 6

Titering the Cosmid Packaging Reaction.......................................................................................... 7

Appendix: Host Strain Preparation .................................................................................................. 8

Troubleshooting .................................................................................................................................. 9

Preparation of Media and Reagents .................................................................................................. 9

References .......................................................................................................................................... 11

Endnotes ............................................................................................................................................. 11

MSDS Information ............................................................................................................................ 11

Gigapack III Gold Packaging Extract, Gigapack III Plus
Packaging Extract, and Gigapack III XL Packaging Extract

M

ATERIALS PROVIDED

Quantity

Catalog

Materials provided

Gigapack III Gold-4 packaging extract (blue tube)a 4 × 25 μl — —

Gigapack III Gold-7 packaging extract (blue tube)a — 7 × 25 μl —

Gigapack III Gold-11 packaging extract (blue tube)a — — 11 × 25 μl

λcI857 Sam7 wild-type lambda control DNAb 1.05 μg 1.05 μg 1.05 μg

VCS257 host strainc 1 ml 1 ml 1 ml

Catalog

Gigapack III Plus-4 packaging extract (blue tube)a 4 × 25 μl — —

Gigapack III Plus-7 packaging extract (blue tube)a — 7 × 25 μl —

Gigapack III Plus-11 packaging extract (blue tube)a — — 11 × 25 μl

λcI857 Sam7 wild-type lambda control DNAb 1.05 μg 1.05 μg 1.05 μg

VCS257 host strainc 1 ml 1 ml 1 ml

Catalog

Gigapack III XL-4 packaging extract (red-orange tube)a 4 × 25 μl — —

Gigapack III XL-7 packaging extract (red-orange tube)a — 7 × 25 μl —

Gigapack III XL-11 packaging extract (red-orange tube)a — — 11 × 25 μl

λcI857 Sam7 wild-type lambda control DNAb 1.05 μg 1.05 μg 1.05 μg

VCS257 host strainc 1 ml 1 ml 1 ml

a

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.

b

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

c

The VCS257 host strain, included for plating the λcI857 Sam7 positive control, is shipped as a frozen bacterial glycerol

stock (see Preparation of 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.

#200201

#200204

#200207

Catalog
#200202

Catalog
#200205

Catalog
#200208

Catalog
#200203

Catalog
#200206

Catalog
#200209

STORAGE CONDITIONS

Packaging Extracts: –80°C (Do not allow the packaging extracts thaw! Do not store the packaging

extracts in liquid nitrogen as the tubes may explode.)

Lambda Control DNA: –80°C (Store at 4°C after thawing.)

Bacterial Glycerol Stock: –80°C

Revision B © Agilent Technologies, Inc. 2010.

Gigapack

III Packaging Extract 1

INTRODUCTION

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.

1-4

Because choice of an in vitro packaging extract designed to achieve specific
experimental goals is important, we offers three different packaging
extracts: Gigapack III Gold packaging extract, Gigapack III Plus packaging
extract, and Gigapack III XL packaging extract. Gigapack III Gold
packaging extract produces the highest packaging efficiency commercially

9

available (2 × 10

pfu/μg) and is designed for use in cDNA and genomic

library construction. Gigapack III Plus packaging extract is an economical
alternative to Gigapack III Gold packaging extract when the highest
packaging efficiency is not required. Gigapack III XL packaging extract
preferentially packages large inserts (i.e., 47–51-kb recombinants), which
eliminates the need for time-consuming size fractionation steps and the loss
associated with sizing columns or sucrose gradients. This packaging extract
is designed for use in genomic lambda and cosmid library construction.

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 plaque­forming 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

9

1 × 10

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

However, predigested arms, when ligated to a test insert, yield

6

~5 × 10

– 1 × 107 recombinant plaques/μg of vector.

2 Gigapack III Packaging Extract