SOUTHERN [MM1] BLOTTING ON GENOMIC DNA

11/5/98 MGM

 

1.      DIGEST DNA

 

Begin with genomic DNA resuspended in TE.� Use wide orifice tips at all times for handling DNA.� It is VERY gooey

 

Digest DNA overnight, and remember that it is important to use lots of enzyme (prefer really robust enzymes) since you are digesting lots of DNA here.� I set� the reaction up in 50 ml.� Make up a master mix containing enough of everything (except DNA) for all of your digests (plus a little extra for pipetting error), then add the DNA to the labeled tubes and add 30 ml master mix to each tube.

 

DNA��������� ����� 20 ml

Buffer�������� ����� 5

BSA (if nec.)����� 0.5

Enzyme����� ����� 5

dH20��������� ����� 10

 

 

2.      RUN AND TRANSFER GEL

  1. Run a medium-sized gel with large-toothed combs.� 0.8% is what you should typically run.� I find about 125-150 ml of agarose per gel is about right.� Add 5 ml per digest of loading dye and load all 50 ml of the digests onto the gel-- you can use normal tips now.� Use a marker, but put it on the end, so you can cut it off (marker will non-specifically bind many probes).� Run the gel 12-18 hours at 25 V constant voltage.� Running slowly like this is better, since it improves and reduces the chances of melting your gel.
  2. Photograph the gel.� Then put it in a tray with enough denaturation solution:

0.5 M NaOH

1.5 M NaCl

to cover the gel.� Rock on rocker for 45 minutes to one hour.� Pour of denaturation solution and rinse with deionized water 2-3 times, then add enough neutralization solution:

1 M Tris pH 7.4

1.5 M NaCl

to cover and return to rocker for one to one and a half hours.

  1. Set up transfer:� Take a pack of paper towels, divide in half and lay the stacks side by side on a plexi surface.� You can transfer either one or two gels on top of this.� Place 5 pieces of gelblot paper for each gel on top of paper towels (either in the middle for one, or side-by side for two gels).� Cut Hybond (transfer membrane-WEAR GLOVES) to the correct size and wet along with a piece of gelblot paper in 10X SSC.�

(10X SSC is diluted from 20X SSC):

For 1 liter (20X)-����������� 175.3 g NaCl

����������� ����������� ����������� 88.2 g Na Citrate

����������� ����������� ����������� pH to 7.0 with small amt (2 drops+/-) HCl

Place wet gelblot and Hybond on top of stack, smooth out air bubbles.� Place gel on top of hybond, again smooth out bubbles.� Wet 2 more pieces of gelblot paper with 10X SSC and place on top of gel.� Cut wick from Whatman (same width as the 1 or 2 gels), wet in 10X SSC.� Place one end of wick on gel setup, the other in reservoir of 10X SSC.� Reservoir shold be high enough that wick will not touch paper towels, etc..� Place flat, waterproof sfc on top of gel setup (e.g. plexi board) and place heavy weight on top (e.g. Sigma catalogue).� Transfer overnight.

  1. In AM take off the wick and top gelblot paper.� Use a pencil (NOT a pen) to mark the wells on membrane.� Place damp membrane on Whatman paper and crosslink� (DNA side up) in Kahn UV crosslinker.� Put membrane in dH2O and add a small amt of EtBr for 20 minutes.� Destain 2 x 20 minutes with dH2O.� View with UV handlamp, mark markers with pencil and use razor blade to remove marker lane.� Return to dH2O until ready to prehyb.

 

3.      PREHYBE AND PROBE MEMBRANE

  1. Make prehyb.� If you are probing etc in hyb oven, you need 100 ml per probe (50 ml to prehyb in and the other 50 for probe later).

100ml--����������� 30 ml 20X SSC

����������� ����������� 50 ml formamide (kept at 4 C)

����������� ����������� �5 ml 100X Denhardt's (kept at -20)

����������� ����������� 10 ml dH20

����������� ����������� �5 ml 10% SDS (ADD THIS LAST).

Sterile filter the solution and split into 2 x 50 cc tubes.�

Heat HS or SS DNA (10mg/ml) (in freezer) in heat block for 5 minutes, ice for 5 minutes.� Add 0.5 ml of the heated/iced HS or SS DNA to one of the 50 ml tubes of prehyb and add to membrane in hyb cylinder.� Place in hyb oven and prehyb 4-6 hours at 42o C.

  1. Make Probe.� Use NEBlot kit.� Requires 25-75 ng DNA.� If unsure of your concntration, try several amounts.� Best probes are restriction fragments or PCR products that have been run on gel and genecleaned.�

1.      Bring probe DNA to 33 ml with dH2O.� Boil 5 minutes, ice 5 minutes.

2.      To DNA, add 2 ml each of dATP, dGTP, dTTP (NO dCTP).

3.      Add 5 ml buffer

4.      Go to hot area.� Add 5 ml fresh a(32P)-dCTP.� Add 1 ml Klenow.� Put in lucite 37o C block and put in incubator.� After 30 minutes, add additional Klenow and incubate another 30 minutes.

5.      Prepare NICK column (Pharmacia) by pouring out top buffer and rinsing with TE.� Fill up column with TE and unplug the bottom to let it drip through.� When the reaction is done, add it to the top of the NICK column, let sit 3-5 minutes.� Add 400 ml TE, let run in.� Add another 400 ml TE and collect what comes out of the column to KEEP- THIS IS PROBE.� Count 1 ml of probe in 6 ml scint fluid.� Should be close to 400,000 cpm.� No less than 250-300,000 cpm.� Probe can be frozen and is OK for a week or so.

  1. Probe blot overnight:

Boil and ice (separately) probe and SS or HS DNA.� Add SS/HS to remaining 50 ml prehyb, mix, add Probe, mix, add to membrane and prehyb in cylinder and return to hyb oven overnight 42o C.

  1. Wash blots:

Pour off probe solution into speedi-dry.� Wash membranes in cylinder/hyb oven 3 x 20 minutes in 50-75 ml per wash of:

2X SSC, 0.5% SDS.

 

Dry membranes, expose to autoradiography.

 [MM1]