Long PCR stuck in the wells :-( (fwd)

Dan Diaz bl275 at cleveland.Freenet.Edu
Wed Jun 8 22:46:54 EST 1994


In a previous article, rapr at MED.PITT.EDU (Robert Preston) says:

>> We are trying inverse PCR of 6 kb plasmid according to Barnes' mixing polymerases
>> strategy. Some primer sets worked real well, while others not so well. However, 1
>> set of primers produced either nothing or heavy EtBr band stuck in the well.
>> 
>> Anybody has any clue as what the stuff is and how to "move" it down to the right
>> size PCR band?
>> 
>Hong, Barnes himself posted (search the archives) that too many cycles
>makes for junk in the gel wells: he said to cut it to 16 cycles to avoid
>that.  I have no idea why, myself.

try using 10X dye with some SDS, as you may have protein-DNA aggregates.  I
have done up to 30 cycles of long-range PCR without problems.

diz



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