1) Need to cut 10 µg of plasmid in two spots ('A' and 'B') in polylinker. 'A'cut is 'ith a restriction enzyme which gives a 3' recessed end (exonuclease sensitive) next to insert. Since 3' recessed end can be relative to coding or noncoding strand, 'A' cut can be on either side of insert. [For sequencing both strands, will need to make nested deletions from both sides.] After a 1 hr (100 µl) 'A' restriction digest 37°C, take a 2.5 µl aliquot for gel analysis, then add 10 µl of 3 M sodium acetate, pH 5.2, and 200 µl of 100% ethanol. Spin for 15 min at 4°C. Remove supernatant. Add 500 µl of 70% ethanol, vortex gently, then spin for 5 min at 4°C. Remove supernatant, add more 70% ethanol, vortex, spin, pull of ethanol, then let DNA pellet air dry for 5 min.
2) Make pellet up in ddH2O and do 'B' restriction digest for 1 hr (37°C) in 100 µl. 'B' cut is with a restriction enzyme which gives a 3' protruding end (three base or greater; exonuclease resistant). Save 2.5 µl of digest for gel analysis.
3) Add 1.35 µl of 1/3 diluted 1 M MgCl2 to give a final concentration of 10 mM Mg. Heat for 10 min at 70°C
4) To titer phage library, thaw phage, and aliquot if it is a newly received library. Add 3 µl of 10 fold dilutions of phage (undil. to 1/2000; dilute in 10 mM MgSO4; best dilution for #1055 in Exp.1727 was 1/1000 - 1/1333) to 600 µl of diluted cells (from step 3)) in sterile blue top (#14-956-4A) tubes. Mix by shaking or gently vortexing, then incubate for 15 min at 37°C on a shaker. During this time, melt top agarose and have cooling to 47°C (check temperature on outside of bottle with BioRad temp. indicator strip).
5) Add 6.5 ml of 47°C top agarose (keep top agarose in a 50°C water bath on the bench by the flame to keep from solidifying) to the first tube, mix by inversion several times, then quickly pour onto the center of a 150 mm NZY plate. Quickly swirl to promote even distribution. Repeat with the next tubes. After all plates have been poured and hardened, invert and incubate at 37°C. Count # of plaques after 12-16 hrs.
6) Using same host cells from 3), dilute phage to give 15,000 - 20,000 cfu/150 mm plate. Do 20 plates, as per 4) and 5). Incubate 12-16 hrs at 37°C.
7) Put plates at 4°C for 2 hrs. During this time, prepare solutions to be used in steps 7) - 9), including prehybridization and hybridization solutions which should be placed in a 42°C incubator to warm. Label first 137 mm filter (Stratagene #420107) with fine tip indelible marker to correspond to plate. Holding filter at edge with blunt ended forceps, place filter onto plaque surface. Mark filter in 3 asymmetric locations by stabbing through it and agar with 18 gauge needle attached to a syringe containing waterproof black ink. After 1 min, pull off filter and immerse DNA side up for 1-5 min in a glass tray containing 500 ml of 0.5 N NaOH, 1.5 M NaCl (25 ml of 10 N NaOH and 150 ml of 5 M NaCl made up to 500 ml in ddH2O). After 10 filters, will need replace this with fresh denaturing solution.
8) Remove and transfer to neutralizing solution for 5 min consisting of 500 ml of 1.5 M NaCl, 0.5 M Tris, pH 7.4 (150 ml of 5 M NaCl and 250 ml of 1 M Tris, pH 7.4 made up to 500 ml in ddH2O).
9) Rinse filter in two successive 2 x SSC baths (400 ml each; 80 ml of 20 x SSC made up to 800 ml in ddH2O), then place DNA side up on fresh 3MM Whatman or bench top paper to dry (1 hr). When all filters are dry, sandwich each between 3MM Whatman paper, and bake at 80°C for 2 hrs under vacuum (need dry ice in trap).
10) Allow filters to cool, then individually wet in 2 x SSC (in a 150 mm petri dish; need about 50 ml), followed by immersion in prewarmed prehybridization solution contained also in a 150 mm petri dish supported in a plastic box. Put top on box, and put in incubator at 42°C for 2 hrs with gentle swirling. After prehyb, individually transfer filters into a 150 mm petri dish (in plastic box) containing hybridization solution. Place top on box and put in incubator at 42°C overnight with gentle swirling.
Prehybridization Sol'n Hybridization Sol'n ddH2O 15.3 ml ddH2O 15.3 ml Formamide 15 ml Formamide 15 ml 20 x SSPE 12.5 ml 20 x SSPE 12.5 ml 20% SDS 0.25 ml 20% SDS 0.25 ml 50 x Denhardt's 5 ml 50 x Denhardt's 5 ml Salmon Sp. DNA 2 ml Salmon Sp. DNA 2 ml Poly A 5 µl Poly A 5 µl 32cDNA probe 50-100 µl
(note: Salmon Sperm DNA stock is 5 mg/ml; Poly A stock is 10 mg/ml; make up prehyb and hyb solÕns in 50 ml conicals) 11) Remove filters individually into 400 ml of room temperature 2 x SSC, 0.1% SDS (40 ml of 20 x SSC and 4 ml of 20% SDS made up to 800 ml in ddH2O). Wash for 5 min on rotator. Do two additional washes. Transfer filters to 400 ml of prewarmed (42 - 65°C; use 42°C for wheat-mouse and 65°C for mouse-mouse hybridizations) 0.1 x SSC, 0.1% SDS (4 ml of 20 x SSC and 4 ml of 20% SDS made up to 800 ml in ddH2O). Wash for 20 min on rotator. Do a total of three 20 min washes.
12) Allow filters to dry on fresh bench paper, then place on Whatmann 3MM paper in a large x-ray cassette holder. Cover with plastic wrap. Under red light in the dark room, place x-ray film on, followed by an enhancing screen. Close up and expose overnight at -70°C. The next day develop film. Copy location of positive plaques and three asymmetric ink marks onto a clear acetate sheet. Place acetate sheet on bottom of original 150 mm plate and identify positive plaques.
13) Pick positive plaques by stabbing with a plastic transfer pipet, and squirt into 1 ml of SM containing a drop of chloroform. Let sit for 1-2 hrs at RT or overnight at 4°C. Vortex gently. Dilute 1/100 (2 µl up to 200 µl) in 10 mM MgSO4. Add 50 µl of diluted sample with 600 µl of XLI-Blue MRF' host cells (OD600=0.5), and mix, then incubate for 15 min at 37°C on a shaker. Add 6.5 ml of 47°C top agarose, mix by inversion several times, then quickly pour onto the center of a 150 mm NZY plate. Quickly swirl to promote even distribution. Incubate at 37°C. Repeat steps 7) - 12). This is the 2° screen. 14) For the tertiary screen, pick positive plaques, as in 13). Dilute 1/1500 so that plaques will be well separated. Mix with host cells as above and plate with top agarose. Repeat steps 7) - 12). All plaques should now be positive. Pick a well-separated plaque from each of the tertiary screen clones. Place in 500 µl of SM containing 20 µl of chloroform, vortex gently and incubate for 2 hr at RT, or store at 4°C. Go to ÔExcision of Clones as pBluescript from lZAPII'. NOTE: to do excision experiment on same day as tertiary plaque picking, need to have prepared overnight cultures of XLI Blue MRF' and SOLR (established from fresh streaked plates).
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