Southern Blotting
4/14/04
Cut 10-20ug total genomic DNA overnight with the appropriate restriction enzyme. Ensure there is adequate enzyme for the cut (check the units/mL – 1U cuts 1ug in 1 hour – most enzymes are 3-20U/uL), and spec DNA to calculate concentration. Total volume needs to be less than 25uL for loading. For early tests of a new enzyme or DNA prep, also run uncut DNA. For ES cells 10ug is about the total DNA from 1 well of a 24 well plate, for tails it is about 1/2 of what you get from 1 tail. For southerns on tails, it is imperative to preserve the tail biopsy in 70% EtOH prior to genomic DNA preparation in order to prevent DNAse digestion during the proteinase K treatment step.
Add 6X sample buffer and load on 0.7% TAE agarose gel with Ethidium. We make the gels with SeaKem Gold agarose for pulsed-field gel electrophoresis (Cambrex cat #50152) as it holds together best at low percentages.
Run the gel slowly enough that it takes about 3-4 hours (will help keep the bands sharp). I use lambda HindIII markers from NEB for molecular weights 1-20kB.
UV photograph the gel with a ruler. This will allow you to localize the markers and will ensure that there is equal loading of DNA. You should see a big smear.
Wash 10 min. 0.2N Hcl in a big baking pan, slowly shaking.
Quick wash with ddH2O
Denaturation solution 2 x 15 min.
Quick wash with ddH2O
30 minute wash with neutralization solution
Transfer the DNA to Hybond N membrane in 10X SSC 4 hours to overnight using a Turbo Blotter or other transfer set up.
Mark the lanes with a pencil, then dissemble the transfer and UV crosslink DNA to the blot (in our not so fancy box, it is 4Õ for crosslink).
PROBE
1. PCR or digest probe sequence 500-1000 bp, you'll need 25-50ng per probe
2. Mix 25-50ng DNA into TE for a final volume of 45µL
3. Denature 95-100¡C for 2-3 minutes.
4. Cool on ice for 2 minutes
5. Centrifuge briefly
6. Get a Amersham RediPrime II tube RPN1633
7. Make sure blue pellet is at the bottom of the tube.
8. Add the 45µL denatured DNA and dissolve pellet. Pipette carefully to avoid bubbles.
9. Add 5µL fresh (preferably) 32P-dCTP (NEN, Easytides, 50µCi)
10. dCTP is green, pipette to make uniform solution.
11. Place in radioactive box in warm room (37¡) for 5-30 mintues (I've been doing 30).
12. Prepare G50 column (Amersham Probequant) by vortexing to resuspend the resin (not the white band). Slightly open and snap off the bottom of the column (snap HARD at score)
13. Place tube in open screw cap tube and spin 3K, 1' to clear column. Dispose of tube, move column to new tube.
14. Add the probe to the top of the column. Spin 3K, 2'. Green color should stay behind.
15. Dispose of column, put screw cap on the tube.
16. Remove 1µL of the probe and place pipette tip in small scintialltion vial. You do not need to add scintillation fluid. Count in the scint. counter using the user 10 card and program. Do "count single rack." You expect 200000-400000 counts for that 1 µL.
17. BOIL THE PROBE 5' at 95-100¡C, cool on ice 3-5'.
CHURCH-GILBERT BLOTTING
1. will need to prehyb the blot in 15mL hybridization solution at 65¡C in rotator over for 90min. Bottles for hybridization are on sink nearest tc room, prepare by washing several X in ddH20. These are stored in count off.
2. Add 2x1,000,000 cpm/mL hyb solution in 10mL that is 2x10,000,000.
3. Hybridize at 65¡C O/N
4. Dispose of probe in radioactive waste
5. Rinse blot briefly with 50mL wash solution heated to 65¡ and dump down sink (record!)
6. Wash 3X 30' at 65¡ in rotator oven.
7. Wrap blot in saran wrap and expose to PI screen (2hrs - O/N) or film with screen at -80¡C for O/N - several days.
20X SSC:
in 400 mL ddH2O:
87.65 g NaCl
44.1g solium citrate (or 50.25g Na citrate 2H2O)
adjust pH to 7.0 with a few citric acid crystals.
adjust V to 500mL
0.2N HCl
490mL ddH2O
8.6mL concentrated HCl
87.75 g NaCl
20g NaOH
water to 1L
87.66g NaCl
6.7g Tris base
70.2g Tris Hcl
water to 1L
Church-Gilbert Hybridization solution:
for 50mL:
7.4mL ddH2O
25mL 1M NaHPO4 pH 7.2
17.5 mL 20% SDS
0.1mL 0.5M EDTA pH 8.0
0.5g BSA (Sigma 7906)
Church-Gilbert Wash Solution:
for 500mL:
450mL ddH2O
20mL 1M NaHPO4 pH 7.2
25mL 20% SDS
1mL 0.5M EDTA pH 8.0