Screening lambda phage
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
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
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.
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
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,
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.
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
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
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.
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.