1) Prepare genomic DNA (ref. p. 9.22, Maniatis) from cultured cells using one T75 flask (wash two times with PBS and then use a rubber policeman to release), or cells from frozen stock. Wash the cell pellets two times in ice cold PBS, and depending on size of pellet make up in 300 - 600 µl of TE, pH 8. Add 7.5 volumes of 6 M guanidine HCl/O.1 M NaAcetate (17.2 gm GuHCl and 1 ml of 3 M NaAcetate made up in 30 ml of ddH2O). Mix on rotator for 1 hr at RT. Prepare a 50 ml conical containing 18 ml of RT 100% ethanol. Using a 5 ml plastic pipet, carefully layer the guanidine HCl dissolved cell pellet below the ethanol.
2) Using a bunsen burner, prepare a glass pipet with a looped and sealed end. Place looped pipet into the ethanol and stir the interface until completely dispersed. The genomic DNA collects on the loop. Wash by transferring to a 10 ml conical containing 5 ml EtOH, and then to another 10 ml conical with 5 ml of EtOH. Allow the excess EtOH to drip off (can touch to a Kimwipe to remove but don't let it over dry) and immerse in 1 ml of TE, pH 8 overnight at 4°C. Free the DNA by gently scraping with fresh sealed pipets. Remove the pipets and rotate at 4°C (24-48 hrs or longer) until the DNA has dissolved. Determine DNA concentration with Hoefer minifluorometer (see Lacrimal Isolation in Lab Protocols). Note that handling genomic DNA requires: (i) use of tips whose bore size has been increased by cutting off the end with a sterile scalpel blade, (ii) for digestions, allow DNA to stand at 4°C for several hours after dilution and addition of 10 x restriction enzyme buffer), (iii) no vortexing (mix with sealed end of glass pipet while keeping mixture at 4°C, particularly after addition of restriction enzyme), and (iv) after digestion for 30 min, add a second aliquot of restriction enzyme and stir.
3) Digest 10 µg of genomic DNA overnight with Eco RI, Hind III or Bam HI. After digestion, concentrate by EtOH precipitation, remove all traces of EtOH by heating for 10 min at 70°C, and make up the pellet in gel loading buffer. In control digests, include 10 µg genomic DNA plus 10-5, 10-6, 10-7 µg of plasmid containing insert complementary to probe. Load on far side of gel away from test samples. As usual, include size standards. Use a 0.7% agarose mid-size gel in 0.5 x TBE containing EtBr. Run overnight at 35 V (constant). Similarly for Southerns for ordering newly identified cDNA clones, restriction cut the miniplasmid prep with an enzyme which should remove the insert (ie. Eco RI). Run on gel, as above. If DNA has been stored at 4°C, heat to 56°C before applying to the gel.
4) After electrophoresis, photograph on transilluminator with florescent ruler. Cut off the left bottom corner to aid in orientation. Transfer is carried out exactly as in Northerns. Transfer gel to a clean glass tray and immediately denatured in 900 ml of 50 mM NaOH/10 mM NaCl (to 893.7 ml of ddH2O add 4.5 ml of 10 N NaOH and 1.8 ml of 5 M NaCl) for 20 min on a rotator. Carefully pour off and add 900 ml of 0.1 M Tris, pH 7.6 to neutralize (20 min on rotator). Pour off and add 900 ml of 20 x SSC for 45 min with gentle rotation. To keep gel immersed during above steps, can balance an inverted ddH2O filled 50 ml conical on it, making sure that the top surface of the gel is wet before placing on the conical.
5) During the above 20 and 45 min steps, preparations for the overnight transfer can be initiated. Needed are: (i) a glass tray or lucite box filled to a depth of 2 cm with 10 x SSC, (ii) a lucite support for the gel (can invert the gel mold in the reservoir), (iii) a long piece of 3 MM paper which stretches over the lucite gel support on either side into the 10 x SSC; wet with 10 x SSC, (iv) nitrocellulose (Amersham Hybond ECL) cut to the same size as the gel (wear gloves in this and all steps), (v) two pieces of 3MM paper cut to the same size as the gel; place one on the long 3 MM piece and wet with 10 x SSC, (vi) a 6-8 inch stack of paper towels cut to the same size as the gel, (vii) a glass tray containing ddH2O, (viii) a glass tray containing 10 x SSC, (ix) two 10Ó x 12Ó pieces of X-ray film, and (x) flat forceps.
6) To set up the transfer, sandwich the gel between the X-ray film pieces and carefully invert (do over the 20 x SSC). Place back side up on the wetted gel-size 3MM paper (on inverted mold). Remove any air bubbles by gently rolling a capless 50 ml conical over the gel. Add some 10 X SSC to keep from drying out. Completely surround the gel with Parafilm or plastic wrap. Next, wet the nitrocellulose paper by floating on, then immersing in, the ddH2O. Immerse the wet filter in 10 x SSC for 5 min. Holding the filter by two sides, carefully set down exactly on the gel (can't reposition). Carefully roll the capless conical over the filter to ensure removal of any air bubbles. Add the remaining gel-size piece of 3MM paper (wet with 10 x SSC) and roll with conical. Carefully add the cut paper towels to a height of 6'. Balance a glass tray on top of the towels to weigh down; stabilize the tray with tape running down on all sides to the 10 x SSC reservoir. Cover with plastic to reduce loss to evaporation. If transfer is initiated in the morning, carefully change the paper towels (can reuse the top dry ones) before leaving at the end of the day. Let the transfer go overnight.
7) Have 80°C vacuum oven heating up. Remove the paper towels and, with the gel/filter still in place, invert. Use an indelible fine tip marker to mark on the filter the edges of the gel (including the cut corner) and the loading wells. Record experimental number at the top. Peel off the gel and stain for 45 min in 0.5 µg/ml ethidium bromide (then place in ddH2O and examine/photograph on transilluminator). Place filter in 6 x SSC for 5 min to wash, then place on 3MM paper and dry for at least 30 min. Place the dried filter between two pieces of 3MM paper and bake under vacuum for 1-2 hr.
8) To carry out hybridization to filter, prehybridize filter for 4 hr at 42°C (in prehyb/hyb. plastic boxes; gently rotation) in a prewarmed solution of 30% formamide, 5 x SSPE, 0.1% SDS, 5 x Denhardt's, 0.2 mg/ml sonicated denatured salmon sperm DNA'
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 sol'ns in 50 ml conicals)
9) During prehybridization, label cDNA probe (purified insert) by random priming method (Pharmacia #27-9251-01). Reconstitute reaction mix by adding 20 µl of ddH2O and place on ice for 5-60 min. Make 1.8 µl of insert up in 25 µl of TE, pH 8 and denature for 3 min at 95°C. Cool on ice for 2 min, then add diluted/denatured insert to reaction mix. Behind lucite, add 5 µl of 32PdCTP (ICN #33004X). Carefully mix by pipetting up and down. Incubate at 37°C for 15-30 min. Purify probe using a Nick Spin Column (Pharmacia #17-0862-02) or equivalent (Qiagen #28304) and check cpm's in Scintillation counter. Denature probe (3 min at 95°C, on ice for 2 min) prior to addition to hybridization solution. Hybridize overnight at 42°C with gently rotation.
10) Remove filter 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 filter 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.
11) Allow filter 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.
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