DNA sequencing - Problem with silver staining (Sep/25/2008 )

hi....
i have a big problem in sequencing.
am trying out manual sequencing followed by silver staining method.
i got the sequencing kit from promega.
i prepared 6% denaturing gel as given in molecular cloning.
once my PCR is over i added stop sequencing solution
while adding the sample to the well it doesnt go into the well. its floating. i tried thrice. all the three time am facing the same problem.
kindly help me
thanks in advance

-Methylase-

QUOTE (Methylase @ Sep 25 2008, 10:57 PM)

In the absence of your complete protocol, I can only ask questions. Please excuse me if they cover 'obvious' steps...
1. When you make the gel, do you wash out the wells before the run?
2. Do you do a pre-run for ~1/2 an hr?
3. Does the stop solution have glycerol etc, to make it denser than the running buffer?

-swanny-

When samples "float" in the wells of a polyacrylamide (assuming your are doing old fashion way and doing your own gels) it could be cause by several factors:
1. there is unpolymerized residue of acrylamide, that's why swanny ask if you wash the wells. If you don't wash them take a syringe and some running buffer or water and add to each well. The syringe is better than the tips because the dispensing is with more force and help to dislodge the residues. Do it carefully so don't bend the walls of the well.
2. not enough glycerol (sucrose or any other reagent) that adds "weight" to the sample. I used to add a little bit more of glycerol always (30-50%).
3. use the tips for gel's (longer and flatter than regular ) so you can introduce it between the plates and make sure the samples is more near the well bottom.

-merlav-

hi,
thanks for ur reply.
the mistake i did is that i didnt wash d wells. yesterday i repeated the same process but washed the gel with buffer and gave a prerun for 30 min.
it worked out well.....
thank u

-Methylase-