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]