Southern Blotting
1. Use re-suspended DNA to make a cocktail of:
10 ml DNA
2 ml Enzyme
5 ml 10X Buffer
33 ml ddH2O
______
50 ml total volume/tube
(can make a master mix of enzyme, ddH2O, and 10X buffer)
2. Incubate at 37°C for 6-8 hours, or overnight
3. Prepare a 0.8% agarose gel for up to 30 samples:
30 mL of 10X TAE
270 ml dH2O
2.5 g agarose
Prepare a 1% agarose gel for up to 60 samples:
30 mL of 10X TAE
270 ml dH2O
3.0 g agarose
Heat in microwave for ~3 min. and add 10ul Ethidium Bromide once slightly cooled. Pour gel once flask is comfortable to touch (not too hot). Make gel at least ~2 hrs before starting.
4. Use 2 L of 1x TAE buffer to fill chamber. Add 10 ml EtBr to buffer.
5. Add 5 ml of Orange G loading dye to each digest tube. Load gel with 30 ml (for thin combs, 20 well per row) of cocktail from each tube, as well as 10 ml of standard (BsteII). Run overnight at 32 volts/ 25 millliamps.
6. The next day, take a picture of the gel using fluorescent ruler as guide, lined up with wells of gel, then rinse gel in 1 liter of Denaturing Solution for ~1 hour (can make and store denaturing solution in advance)
50 mL NaOH (10N)
303 mL NaCl (5M)
______
Fill to 1 liter with dH20
7. Rinse gel twice with dH20
8. Wash gel with 1 liter of Neutralizing Solution for 1- 1 1/2 hours (make fresh neutralizing solution!)
NaCl 300mL (5M)
Tris 121.14 g
pH to 7.0 with HCl
______
Fill to 1 liter with dH20
9. While gel is still in the neutralizing solution, put 2 layers of Whatman 3M paper around a glass plate to form a support for the gel. Place the support inside a large dish filled with 20X SSC, wetting it as you bring it over the glass plate. When it is completely wet, smooth out all bubbles.
10. Slide another glass plate under the gel to remove it from the neutralization solution and flip it upside down onto the Whatman paper. Again, make sure that there are NO AIR BUBBLES!!
11. Surround the gel with Saran wrap
12. Cut a piece of nitrocellulose paper the approximate size of the gel, soak it first dH20, then in 20X SSC. Then transfer the membrane onto the gel (do not touch membrane with your hands, and do not move membrane once it has been put onto the gel!) Smooth out any air bubbles!
13. Cut two pieces of Whatman paper similar in size to the gel, wet them with dH2O and then with 20X SSC, place them onto the membrane, and smooth out any air bubbles!
14. Cover the Whatman paper with a thick layer of paper towels (stacked and staggered, about 3 inches thick), put a glass plate on top, and weigh down (either with solution bottle or heavy catalog; 500 g weight). Allow transfer to occur O/N. Can even put fresh paper towels next morning and transfer until afternoon.
15. The next day, remove paper towels and Whatman paper above the gel. Turn over the gel and membrane and lay them, gel side up, on a dry sheet of 3M paper. Mark the wells of the gel on the membrane, then remove and discard the gel.
16. Bake the gel for 2 hours at 80°C in the vacuum oven. At this point, the membrane can be stored until ready to use.
17. Prepare the probe using the Megaprime Labeling Protocol
18. When ready to hybridize, roll up membrane and insert into hybridization tube. Fill tube with 30-60 ml non-radioactive hybridization juice and let membrane stick to inside of tube. Place in 42°C oven for 3 hours for pre-hybridization.
19. Boil necessary amount of probe (1 million counts/mL) for 5 minutes, chill on ice for 2 min. Pour non-radioactive hybridization juice back into a 50 ml Falcon tube. Spin down probe, then add to hybridization juice. Pour radioactive hybridization juice back into bottle.
20. Hybridize for overnight at 42°C. Keep in oven until ready to wash.
21. Drain hybridization juice from tube + discard. Remove membrane and wash with:
100mL 20X SSC *Make sure to add dH20 after SSC or
10mL 10% SDS solution will precipitate
______
add dH20 to 1 liter Wash at room temp. for 30-45 minutes
22. Wash a second time with:
25mL 20X SSC
10mL 10% SDS
______
add dH20 to 1 liter and heat in microwave for ~4 minutes
Wash at 55ºC for 30-45 minutes
23. Dry membrane on a piece of Benchkote paper.
24. Wrap membrane with saran wrap
25. Put wrapped membrane on a piece of Whatman paper and check with counter. Expose filter desired length of time, using intensifying screen, at -80ºC.
Updated 8/18/10 by RK