Screening lambda phage libraries


Introduction

cDNA and genomic DNA libraries are useful resources for obtaining fish genes. Fish cDNA libraries are essentially the same as mammalian cDNA libaries and the same concepts apply. A library with a base of about 1 million recombinants is desirable and it may be necessary to screen all of these to be sure of obtaining all genes of interest. In contrast, fish genomic libraries constructed in l phage are considerably more useful than their mammlian derived counterparts. Many fish have relatively small genomes; for example the flatfish genome is only one fifth the size of the mammalian genome and correspondingly the equivalent genes are about one fifth the length. This means that for flatfish only two 20cm dishes need be screened to adequately interrogate the entire genome and the resulting l phage clones are far more likely to contain the entire gene of interest than a clone from a mammalin library.

The best way to screen a library, cDNA or genomic, is with a nucleic acid probe because, assuming hybridisation is occuring one is 5X more likely to obtain a sequence from a cDNA library wirh a nucleic acid probbe than with an antibody. It would be pointless to screen a genomic library with anything other than a nucleic acid probe.

However it is possible to screen cDNA libraries, constructed in suitable vectors, with antibodies and this has proved useful in obtaining genes for which proteins are available and where suitable nucleic acid probes are unavailable.

Plating
 


Plaque lifts

Immunoscreening
 


Nucleic acid probes

Visualisation

Immunological Screening
 


Nucleic acid probes
 


Positive plaque selection
 


Reagents

LB Media; Bactotryptone 10g, Bacto yeast extract 5g, NaCl 5g, per litre, pH 7.5 with NaOH. Autoclave and store in 500ml batches.

LB Agar; 10g Bacto agar per litre LB media, autoclave and store in 500ml batches.

Top agarose; 0.6g of agarose (or agar) per 100ml LB media, autoclave and store in 100ml batches.

IPTG; 1M soln in water, filter sterilised, diluted as required, store in freezer.

Maltose soln.; 4.0g/10ml water, sterile filter.

Amp; 25mg/ml water, sterile filter.

l diluent; 0.01M Tris/HCl, pH 7.4, 0.1M NaCl, 0.01M MgCl2, 0.05% gelatin, autoclave.

Reference

Huynh, T. V., Young, R. A., and Davis, R. W. (1985) Constructing and screening cDNA libraries in lambda gt10 and lambda gt11. In DNA Cloning, vol. 1 (ed. Glover, D. M.), IRL Press, Oxford, Washington DC.