- After you have visualized the gel with ethidium bromide and photographed the gel, use a razor blade to mark DNA bands of the size markers.
- Place the gel in a baking dish and pour enough denaturing solution into the pan to cover the gel ( 300-500 ml ). Allow this to set for 30 min. with occasional gentle agitation.
- Pour off the denaturing solution, fill the pan with ddH2O and pour off immediatly, then pour enough neutralizing solution into the pan to cover the gel. This should set for 30 min. with occasional gentle agitation.
- While the gel is neutralizing, you should cut a piece of cellulose nitrate (BA-85 ) or charged nylon (GENESCREEN or HYBOND-N) to the same size as your gel.
**When you handle membrane filters always wear gloves, and only use a virgin razor blade that has been washed with ethanol. This is to prevent oil spots which will not wet.**You will need 5-6 pieces of Whatman 3MM paper a little larger than your gel , and about 11/2-2 inches of paper toweling. A wick of Whatman 3MM paper will also be needed (2;18"x 9")
- After 30 min., pour off the neutralizing solution and pour in about 500 ml of 5X SSC.
- At this point you are ready to construct the apparatus below.
- First, remove the gel from the SSC (scoop it out with a piece of mylar).
- Then pull the wick through the SSC and wrap back around the glass plate so both ends of the wick are in the buffer. **BE SURE TO REMOVE ANY AIR BUBBLES ** (This can be done by rolling a pipet over the bubble.)
- Prewet the membrane filter by laying it on the wick, Once it turns grey (wetted) you should remove it. If it doesn't wet uniformly, don't use it.
- Place the gel on the wick, being careful not to trap any air bubbles between the wick and the gel.
- Place the prewetted membrane on the gel, remove any air bubbles and trim the membrane/gel so there is no overhang.
- Mask off the rest of the exposed wick with Parafilm.
- Place the 5-6 pieces of 3MM paper on the membrane, and the paper toweling on top of that.
- Top it off with a glass plate and cover the whole thing with plastic wrap.
- Apply preasure to the blot by placing about 1 L of a solution in a bottle on top of the plate.
- Allow to stand like this for 4 hours.
- At end of time disassemble the apparatus and mark the size markers with a Bic ball-point pen.
- UV fix the DNA to the membrane while still dampby exposing the blot, DNA side up, to a germicidal lamp for 2 min. appx. 12 cm from the bulb.
READY TO PROBE