Preparation of Nested Deletions

 

Procedure:

 

1) Need to cut 10 g of plasmid in two spots (A and B) in polylinker.  A cut is with 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 l) A restriction digest 37C, take a 2.5 l aliquot for gel analysis, then add 10 l of 3 M sodium acetate, pH 5.2, and 200 l of 100% ethanol.  Spin for 15 min at 4C.  Remove supernatant.  Add 500 l of 70% ethanol, vortex gently, then spin for 5 min  at 4C.  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 (37C) in 100 l.  B cut is with a restriction enzyme which gives a 3 protruding end (three base or greater; exonuclease resistant).  Save 2.5 l of digest for gel analysis.

 

3) Add 1.35 l of 1/3 diluted 1 M MgCl2 to give a final concentration of 10 mM Mg.  Heat for 10 min at 70C 

 

4)  To titer phage library, thaw phage, and aliquot if it is a newly received library.  Newborn mouse brain lZAPII library is #1055 in white box#11 of -70C freezer.  Add 3 l 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 l 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 37C on a shaker.  During this time, melt top agarose and have cooling to 47C (check temperature on outside of bottle with BioRad temp. indicator strip).

 

5) Add 6.5 ml of 47C top agarose (keep top agarose in a 50C 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 37C.  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 37C.

 

7) Put plates at 4C 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 42C 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 80C 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 42C 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 42C overnight with gentle swirling.

 

 

 

         Prehybridization Soln                     Hybridization Soln

                  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 Denhardts   5 ml                              50 x Denhardts   5 ml

         Salmon Sp. DNA   2 ml                              Salmon Sp. DNA   2 ml

         Poly A                  5 l                              Poly A                  5 l

                                                                        32cDNA probe       50-100 l

 

(note: Salmon Sperm DNA stock is 5 mg/ml; Poly A stock is 10 mg/ml; make up prehyb and hyb solns 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 - 65C; use 42C for wheat-mouse and 65C 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 -70C.  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 4C.  Vortex gently.  Dilute 1/100 (2 l up to 200 l) in 10 mM MgSO4.  Add 50 l of diluted sample with 600 l of XLI-Blue MRF host cells (OD600=0.5), and mix, then incubate for 15 min at 37C on a shaker.  Add 6.5 ml of 47C 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 37C.  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 l of SM containing 20 l of chloroform, vortex gently and incubate for 2 hr at RT, or store at 4C.  Go to Excision 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).