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
 

• Melt enough top agarose (7.5ml/140mm plate; 20ml per 20cm dish) in microwave and leave to cool to 48°C in water bath.
  
  
• Add to sterile tube, per plate, 600µl (1.5ml for 20cm dish) Y1090 (or XL1-Blue) plating bacteria (grown overnight in LB, 0.2% maltose, 10mM MgCl2), 20000-40000pfu (100000pfu for 20cm plates) lgt11 (or lZAP) in l diluent. Shake gently at 37°C for 20 mins to adsorb phage.
  
  
• Split adsorption mixture between sterile tubes and add 7.5ml (20ml for 20cm dish) of top agarose at 48°C, with a warmed pipette, to each tube. Mix by 3X inversion and pour onto LB agar plates, pre-warmed to 42°C. Allow to set and then incubate at 42°C (37°C for lZAP) for 2-2.5 hours, until pinhole size plaques appear. They must be densely packed but not overlap. Plaques can be difficult to see at this stage.

Plaque lifts

Immunoscreening
 

• While plates are incubating, soak 140mm nitrocellulose discs with 25mM IPTG and allow to dry (about 0.5-1 hour). Carefully overlay nitrocellulose filters onto agarose and incubate at 37°C for 5-6 hours.
  
  
• Remove nitrocellulose discs carefully after using 18 gauge needle to spear recognition holes through filter and agar, and mark underside of plate with a pen. Store plate at 4°C, and place filter in TBS, 0.5% gelatin, 0.01% sodium azide and leave at 4°C overnight.
  
  
• Incubate the filters next day in TBS/gelatin/azide for 1 hour, followed by 2-3 hours with primary antibody in TBS/gelatin/azide. Wash 3-4 times in TBS and then incubate with the secondary antibody in TBS for 1 hour at the manufactureres recommended dilution. Wash 3-4 times in TBS.

Nucleic acid probes

• Leave plates to incubate for 6 hours or until plaques are visible and then remove from incubator and leave at room temperature overnight.
  
  
• Cut a nylon filter (PALL-Gelman Biodyne B) to the appropriate size and carefully apply the dry nylon filter to the plate. Using an 18 gauge needle, spear recognition holes through filter and agar, and mark plate underneath with a pen. Leave for 5mins and then peel off. Place, plaque side up, on 3MM blotting paper soaked in 0.4M NaOH, 1.5M NaCl for 5mins and then on 3MM paper soaked in 0.5M Tris, pH 8.0, 1.5M NaCl for 5mins. Then rinse for 20secs in 2X SSC and leave to dry for a few mins on paper towel. Bake for 1 hour at 80°C. Store the plate at 4°C.
  
  
• Prehybridise the filters for 1 hour  (or more) in 10% PEG 8000, 1.5X SSPE, 7%SDS. Add 32P labelled probe and incubate for  between 4 and 20 hours. Hybridisation temperature is dependent on probe sequence and size. Filters are washed 3X in 2XSSC at room temperature and further washed if judged necessary in decreasing concentrations of SSC and increasing temperatures.

Immunological Screening
 

• Rinse filters once for 2 min in 100mM Tris/HCl, pH 9.5, 100mM NaCl, 5mM MgCl2. Stain by adding a few mls of stain solution to each filter. The best way to distinguish positive plaques is to allow all of the plaques to stain faintly and then stop the reaction by several rinses in distilled water. Positives are then distinguished as darker versions of the general plaque background. Staining solution is made up immediately before use by adding 100µl of NBT stock (34mg/ml in 70% DMSO, stored frozen in 1ml aliquots) and 100µl of BCIP stock (17mg/ml in water, stored frozen in 1ml aliquots) to 10ml of 100mM Tris/HCl, pH 9.5, 100mM NaCl, 5mM MgCl2.

Nucleic acid probes
 

• When washing is finished filters are allowed to dry to dampness on paper towels and autoradiographed. Be sure to align the filter in the cassette in such a way as to allow re-presentation of the autorad so that the positions of the alignment holes can be marked.

Positive plaque selection
 

• Positive colonies are located on the plate by placing it on the filter or autoradiograph, plaque side down,  aligning the recognition marks and locating the area of interest. Plug out the positive area using the fat end of a blue pippetteman tip. It is impossible to select a single positive plaque at this stage, and so subsequent screens at lower plating densities are required. Plugs are placed in 1ml of phage storage buffer with 25µl of chloroform. Incubate overnight at 4°C and proceed to repeat the above steps using 0.1-1µl plug soln. instead of l library, with 150µl of plating bacteria on a 90mm LB plate. The first rescreen should have at least 100 plaques to be sure of identifying a positive. Repeat this until single well spaced positive plaques can be picked (2-4 cycles). Then make pure lgt11 phage DNA prep or in vivo subclone from lZAP.

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.