Πgt 11 screening for DNA-binding proteins
A.) Plate and grow phage:
1.) Library is in Πgt 11
2.) The night before grow Y 1090 r-: 20 ml LB-Mg medium
0.2 ml 20% maltose
10 ul 100 mg/ml Ampicillin
100 ul Y 1090 r-
Grow at 37 C O/N.
3.) In am spin at 4 C tablecentrifuge at 3000 rpm for 10min and resuspend in sterile filtered
10 mM MgSO4. Keep on ice.
4.) The night before dry plates at RT under hood ca. 1/2 hour then leave outside at RT O/N.
5.) Prewarm incubator to 42 C.
6.) In am prewarm plates in incubator for 45 - 60min at 42 C.
7.) Melt in MV LB-Mg top agarose 7.5 g/L and cool to 55 C in WB.
8.) For primary screen prepare appropriate dilution to 50000/plate from random primed or oligo dT primed library. 1 : 105 in sterile SM s gelatine.
9.) Add 300 ul of bugs to 15 ml polystyrene tube. Then add approp. amount of phage and shake at 37 C for 20min.
10.) Add 7 - 8 ml of top agarose (55 C) with 10 ml pipette to tube invert once and pour in a conical shape on the plate. Turn plate until completely covered with agarose. Then let plates sit at RT for 30min.
11.) Back to 42 C incubator for ca. 3 hours until plaques are just visible.
12.) Prepare 10 mM IPTG in H2O from 100 mM stock and sterile filter through 0.22 um.
Pour into 150 mm petri dish.
13.) Then label nitrocellulose membranes with pen (remember, when you cut it later, to label it right)and immerse upside down in 10 mM IPTG until wet. Dry on a Whatman.
14.) Take out plates from incubator and overlay with filters upside down. Then back to incubator for another 3 hours at 37 C.
15.) In the meantime nicktranslate probes (see protocol) and purify on a G-50 Sephadex column.
1.) This might increase specific binding of the protein to the DNA, but is only optional. Otherwise proceed with blotting under C.)
2.) Cool plates at 4 C for 10 min. Then mark with ink-needle exact position of filter on the agar.
If filter will be cut for different probe-hyb's, then ink-label asymetrically for as many pieces as needed.
3.) Then lift filters carefully and air dry on Whatman for 10min. at RT.
4.) Then immerse filters (10 filters/50 ml in 150 mm petri dish) in Hepes binding buffer/6 M Guanidine-HCL and incubate at 4 C for 10min. gently shaking. Repeat 1x with fresh Hepes BB/6 M Guanidine-HCL.
5.) Then immerse for 5min. at 4 C in Hepes BB/3 M Guanidine (dilute 6 M-solution with Hepes BB-stock 1:1).
6.) Repeat each time using Hepes BB containing 1:1 dilution of Guanidine-HCL from previous wash.
7.) Then wash 2x in Hepes BB s Guanidine at 4 C. for 5min.
8.) Block in blotto.
1.) If plates are coming from incubator then mark filters with ink needle on agar. Lift carefully and immerse in blotto gently shaking at RT for 1 hour in rubbermaid container filled with 200 - 400 ml blotto. Filters can be cut in several pieces at this point. Make sure, that they are marked well enough.
2.) Then wash filters 2x 1min. in TNE 50 (May need to vary salt concentration) in rubbermaid container. Can cut the filters at this point too, which I would recommend, if there are many of them.
1.) Transfer filters to 50 - 100 ml hybridisation solution. Can be done in petri dish (10/50 ml):
50 ml TNE 50
250 ul Calf Thymus DNA denatured (1 mg/ml)
ul Nicktranslated probe 2 x 106 dpm/ml
2.) Gently shake for 1 hour at RT.
3.) Then transfer to TNE 50 and wash 3x for 10min. at RT. If you have different probes to hyb with, use petri dishes as well for the washings.
4.) Dry filters on Whatman cover with saranwrap and autoradiograph O/N.
E.) Isolation and purification of clones:
1.) In am line up film with filters and mark their position. Then identify positives. They should be in the middle of the plate, not to dark without a dark center. Line up the film with the plates.
2.) Then pick the plaque with the wide end of a sterile pasteur pipette and put into 15 ml polypropylene tube filled with 1 ml SM + gelatin 0.01% and a few drops of chloroform.
3.) Store O/N at 4 C.
4.) In am titer.
5.) For secondary screening plate 1000 - 5000 clones per plate.
6.) Isolate secondaries with small end of a sterile pasteur pipette as above. Should be single clones.
7.) For tertiaries dilute 1/10 in SM and plate 1 or 2 ul. Hyb with a mutant oligo and an unspecific oligo.
1.) TNE 50 20 ml 1 M Tris pH 7.5 10 mM
20 ml 5 M NaCL 50 mM
4 ml 0.5 M EDTA 1 mM
2 ml 1 M DTT 1 mM
1954 ml H2O
Can vary salt to lower stringency for example 10 mM NaCL.
2.) Blotto 10 ml 1 M Tris pH 7.5
2 ml 5 M NaCL
0.4 ml 0.5 M EDTA
0.2 ml 1 M DTT
187.4 ml H2O
10 gm Instant milk
3.) Hepes binding buffer 50 ml 1 M Hepes pH 7.9 25 mM (Sigma cat. H-7006)
50 ml 1 M NaCL 25 mM
10 ml 1 M MgCL2 5 mM
1 ml 1 M DTT 0.5 mM
1890 ml H2O
Make up Hepes BB/6 M Guanidine-HCL ca. 200 ml. Dilute a portion of that with Hepes BB to a 3 M Guanidine-solution. Store at 4 C. Make always fresh.