Southern?Analysis

Southern Analysis

Procedure (for analysis of genomic DNA or ordering of clones):

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 - 600µl of TE, pH 8.Add 7.5 volumes of 6 M guanidine HCl/0.1 M NaAcetate (17.2mg GuHCl and 1 ml of 3 M NaAcetate made up in 30ml of ddH₂O).Mix on rotator for 1 hr at RT.Prepare a 50ml conical containing 18 ml of RT 100% ethanol.Using a 5ml 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 10ml conical containing 5ml EtOH, and then to another 10ml conical with 5ml 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℃.Free the DNA by gently scraping with fresh sealed pipets.Remove the pipets and rotate at 4℃ (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℃ 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℃, particularly after addition of restriction enzyme), and (iv) after digestion for 30min, add a second aliquot of restriction enzyme and stir.

3) Digest 10µg 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℃, and make up the pellet in gel loading buffer.In control digests, include 10µg genomic DNA plus 10-5, 10-6, 10-7µg 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℃, heat to 56℃ before applying to the gel.

4) After electrophoresis, photograph on transilluminator with florescent ruler.Cut off the left bottom corner to aid in ientation.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 4.5ml of 10 N NaOH and 1.8ml of 5 M NaCl) for 20min 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 45min with gentle rotation.To keep gel immersed during above steps, can balance an inverted ddH₂O filled 50ml 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 45min steps, preparations for the overnight transfer can be initiated.Needed are: (i) a glass tray or lucite box filled to a depth of 2cm 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 50ml 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 5min.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℃ 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 45min in 0.5µg/ml ethidium bromide (then place in ddH₂O and examine/photograph on transilluminator).Place filter in 6 x SSC for 5min to wash, then place on 3MM paper and dry for at least 30min.Place the dried filter between two pieces of 3MM paper and bake under vacuum for 1-2hr.

8) To carry out hybridization to filter, prehybridize filter for 4 hr at 42℃(in prehyb/hyb.plastic boxes; gently rotation) in a prewarmed solution of 30% formamide, 5 x SSPE, 0.1% SDS, 5 x Denhardt's, 0.2mg/ml sonicated denatured salmon sperm DNA

Prehybridization Sol'n Hybridization Sol'n ddH₂O 15.3 ml ddH₂O 15.3ml Formamide 15ml Formamide 15ml 20 x SSPE 12.5ml 20 x SSPE 12.5ml 20% SDS 0.25ml 20% SDS 0.25ml 50 x Denhardt's 5 ml 50 x Denhardt's 5ml Salmon Sp.DNA 2ml Salmon Sp.DNA 2 ml Poly A 5µl Poly A  µl 32cDNA probe 50-100µl

(note: Salmon Sperm DNA stock is 5mg/ml; Poly A stock is 10mg/ml; make up prehyb and hyb sol'ns in 50ml 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 5-60min.Make 1.8 µl of insert up in 25µl of TE, pH 8 and denature for 3 min at 95℃.Cool on ice for 2min, 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℃ 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℃, on ice for 2min) prior to addition to hybridization solution.Hybridize overnight at 42℃ with gently rotation.

10) Remove filter into 400ml of room temperature 2 x SSC, 0.1% SDS (40ml of 20 x SSC and 4ml of 20% SDS made up to 800ml in ddH2O).Wash for 5min on rotator.Do two additional washes.Transfer filter to 400ml of prewarmed (42 - 65℃; use 42℃ for wheat-mouse and 65℃ for mouse-mouse hybridizations) 0.1 x SSC, 0.1% SDS (4ml of 20 x SSC and 4ml of 20% SDS made up to 800ml in ddH₂O).Wash for 20min on rotator.Do a total of three 20min 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℃.