Mass Excision of lambda Zap libraries

from the Stratagene Manual

ExAssistª lnterference-Resistant Helper Phage with SOLRª Strain
Catalog #200253


INTRODUCTION

The EXAssistª interference-resistant helper phage with SOLRª strain is designed to allow efficient excision of the BIuescript¨ phagemid from the Lambda ZAP¨ vectors, while preventing the problems that are associated with helper phage co-infection. The ExAssist helper phage contains an amber mutation that prevents replication of the phage genome in a nonsuppressing Escherichia coli strain such as SOLR cells. This allows only the excised phagemid to replicate in the host, removing the possibility of co-infection from the ExAssist helper phage.


MATERIALS PROVIDED

HOST STRAINS

SOLR Strain: e14- (mcrA) D (mcrCB-hsdSMR-mrr) 171 sbcC recB recJ uvrC umuC::Tn5(Kanr) lac gyrA96 reIA1 thi-1 endA1 [F' proAB laclq ZDM15] Su- (nonsuppressing), l'(lambda resistant)

XL1-Blue MRF' Strain: D (mcrA) 183 D (mcrCB-hsdSMR-mrr) 173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F' proAB laclq ZD M15Tn10 (Tetr]

Storage conditions vary. Please see Preparation of Host Cells for details.

HELPER PHAGE

ExAssist interference-resistant helper phage
~1 x 10e10 pfu/ml, single-strand size is 7.3 kb
[comigrates with ~5 kb of double-stranded linear DNA on a 1% (w/v) agarose gel]

On arrival, store at -20 or -80 C. After thawing, aliquot and store at -20 or -80 C.. Do not pass through more than two freeze-thaw cycles. Helper phage can be stored at 4 C for up to 1 month.

NOTE: ExAssist interference-resistant helper phage has a-complementing b-galactosidase sequences which may interfere with sequencing or site-directed mutagenesis where oligonucleotide primers hybridise to b-gaIactosidase sequences (e.g., M13-20 primer). VCSM13 interference-Resistant Helper Phage and R408 Interference-Resistant Helper Phage are recommended for single-stranded rescue.

tetracycline at 12.5 microgrammes/ml.
kanamycin at 50 microgrammes/ml.

On arrival, prepare the following from the bacterial glycerol stock:

NOTE: Do not allow the contents of the vial to thaw. The vials can be stored at -20 or -80 C, but most strains remain viable longer if stored at -80 C.

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

2. Streak the splinters onto a plate containing LB agar (see Media and Solutions) and the appropriate antiblotic.

Restreak the cells fresh each week.

Preparation of a -80 C Bacterial Glycerol Stock

1. In a sterile 50 ml conical tube inoculate 10 ml of appropriate liquid medium containing antibiotic with one or two colonies 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.


GROWTH OF CELLS

Plating cultures should be started from a fresh plate using a single colony and should be grown overnight with vigorous shaking at -20¡ in appropriate medium supplemented with 0.2% (v/v) maftose and 10 mM magnesium sulfate (MgSO4) when required (see the table in Preparation of Host Ceils). (Do not use tetracycline in the presence of magnesium.) The lower temperature ensures that the cells will not overgrow. The cells should be spun at 1000 x g for 10 minutes and then should be gently resuspended in 10 mM MgSO4. Cells prepared In this manner can be used for all manipulations described in this instruction manual. Highest efficiencies are obtained from freshly prepared cells. Before use, dilute the cells to OD600= 1.0 with 10 mM MgSO4.

For further details about phage amplification or tittering, see
Sambrook, J., Fritsch, E.F., and Maniatis, T., eds. (1989)
"Molecular Cloning: A Laboratory Manual," Second Ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.