Southern Blot Analysis
- Digest DNA 5-10 ug overnight with BSA and Sp, completely mix it. Add 1 ul more enzyme the next morning. If the DNA purity is not good or too diluted, precipitate it with isopropanol or ethanol. Then dissolve in 0.5xTE. The DNA concentration should be about 0.5 ug/ul.
- Prepare 400 ml 0.8-1.0% of agarose gel. Load samples (less than 50 ul), then first run the gel at 120 Vlt for 10-15 min, then reverse the polarity and run the gel at 80 vlt for 5-10 min. finally run the gel at 40 vlt for 6-12 hours, then 60 vlt until the end of the electrophoresis. Add bromophenol blue 20 mins before ending in one well.
- Photograph it, cut the gel to 20cm x 15 cm. Invert the gel and dip it into 0.25 M of HCl (23 ml concentrated HCl in 1 L H2O) for 5-15 min (depending the DNA fragment size, usually 10-minute treatment is enough. Treat it until the bromophenol blue become brown??).
- Pour the HCl solution, wash it 2x with ddH2O.
- Add (5L: 100g NaOH, 383g NaCl) for 20-30 mins.
- In a proper tray, put 1 piece of 3 MM paper, then a block of sponge (3 cm thick) on it, pour the 0.5x NaOH/1.5xNaCl solution, remove any bubbles by rolling the sponge in the solution, carefully put 2 pieces of 3 MM paper, one by one, make sure there is no bubble.
- Cut a nylon membrane to 20cm x 15 cm. put it on a metal board to eliminate any static effect. Then dip in 2 x SSC for 5-10 minutes.
- Put the gel on it, then nylon membrane, then 6 (3 wet, 3 dry) pieces of 3 MM paper, and a stack of tissue.
- Put a gel tray on it. Transfer it for 16 hours.
- Wash the membrane in 2x SSC for 3-5 min. Then dry it at 80°C vacuum oven for 2 hours. Keep it properly. (No UV crosslinking!!!)
Probe-labeling:
- Take 25-30 ng of probe DNA (purified from centrifuge miniprep) in 0.5x TE, denature it in boiling water for 10 mins. Immediately freeze it in dry ice/alcohol.
- In a Rediprime II Random Prime labeling tube (RPN1633), add 5 ul of 32P-dCTP (kelow fragment enzyme, ramdom primers), thaw the denatured probe and add it into the reaction tube. 37C for 1-2 hours.
- Purify it with ProbeQuant G-50 Micro Columns (Amersham Biosciences, Cat# 27-5335-01) or NucTrap Probe purification Columns ( Stratagene, Cat# 400701-13).80 ul NTE balance, add 40 ul NTE into reaction tube, transfer it to Column, add another 80 ul NTE to wash it, then a tube of air. Collect the probe.
Hybridization:
- Remove any static effect from the nylon membrane by touch on a metal board. Prehybridize the membrane for 3-5 hours with Church’s buffer :
CHURCH'S BUFFER:
800ml ddH2O
10 g BSA (final 1%)
35.5g Na2HPO4 (final 0.5M)
30.0g NaH2PO4 (final 0.5M)
Mix these into solution. (may require several hours) Adjust pH to 7.0-7.2
2ml 0.5M EDTA (final 0.001M)
70g SDS. (final 7%)
Mix with heat (50°C) to get SDS into solution.
qs. to 1000ml.
20x SSC:
3M NaCl (175g/L)
0.3M Na3Citrate"2H2O (88g/L)
Adjust pH to 7.0 with 1M HCl
or Rapid-hyb buffer (Amersham pharmacia biotech, RPN 1635-125ml 0r 1636-500ml) or QuikHyb (Statagene, Cat# 201220, 250ml). Denature 100-200 ul salm sperm (1 ug/ul) DNA and add it to the prehybridization solution.
Recipe: 6× SSC buffer
20 mM NaH2PO4 (5× Denhardt's reagent)
0.4% (w/v) SDS
500 μg/ml of sonicated or sheared, denatured salmon sperm DNA
(see Prehybridization of the Membrane for Use with Long DNA
Probes)
- Denature the probe (may be done in hybridization tube in boiling water).
- Pour out the prehybridization solution and add 5ml new Rapid-Hyb buffer, warm it up at 65°C for 5-10 mins, add the denaturing probe. Hybridize it overnight.
Washing:
- Washing buffer I (1xSSC, 0.1%SDS), wash twice shortly.
- Washing buffer II (0.1xSSC, 0.1%SDS), wash 3x, 15 min each.
Stripping:
1. Wash the membrane for 30 minutes or overnight at 65°C in the following wash
solution:
50% formamide
6× SSPE buffer or 6× SSC buffer
2. Pour a boiling solution of 0.1× SSC buffer and 0.1% (w/v) SDS or a
boiling solution of 0.1× SSPE buffer and 0.1% (w/v) SDS over the
Southern blot and wash the membrane for 15 minutes (after removing
the membrane from heat). Repeat several times if necessary.
- Wash the membrane for 15 minutes at 65°C in dH2O.
CHURCH BUFFER PREPARATION
Material:
Bovine Serum Albumine Fraction V (BSA), Serva (#11927).
Na2HPO4, 2H2O, Merck (#1.06580.1000)
NaH2PO4, H2O, Merck (#1.06346.1000)
SDS, Roth (#4360,1)
1.Preparation of 1 Liter Phosphate Buffer (0,5 M pH7,2):
Solution A:
Na2HPO4, 2H2O (1M): 177,9 g
di H2O: qsp 1 Liter.
Solution B:
NaH2PO4, H2O (1M): 137,99 g
di H2O: qsp 1 Liter
Mix solution A and B for 1 liter of Sodium Phosphate Buffer (0,5 M pH 7,2) as follows:
Solution A: 342 ml
Solution B: 158 ml
diH2O: 500 ml
Control the pH (pH 7,2).
2.Preparation of 1 liter of Church Buffer:
Final concentration:
-Sodium Phosphate Buffer (0,5M pH 7.2): 500 ml 0,25M
-EDTA (0,5M): 2 ml 1 mM
-BSA: 10 g 1%
-SDS: 70 g 7%
-Dissolve the BSA by small amounts ( per 1 g) in ~50ml distilled water, but do not heat.
-Dissolve separately 70 g SDS in small amounts in ~400 ml diH2O, under stirring and gently heating.
Transfer the Sodium Phosphate Buffer in a 1 Liter measuring cylinder, and add slowly (to avoid precipitation), while stirring, respectively the BSA solution, the SDS solution and the 2 ml EDTA.
Remark: Handle SDS under a hood.
Filtrate on a vacuum filtration unit (Nalgene).
Aliquot by 50 ml.
Store at –20°C.
