primers problem

Tim Myles tmyles at leland.stanford.edu
Thu Feb 15 22:30:51 EST 2001


Hi Tina,

I always resuspend oligos at about 100 µM in TE (pH 8.0) then aliqout and
store at -20°C.

Tim





Tina wrote:

> I study  the expression of TNFa in porcine spleen cells. There is only two
> papers who gives the sequence of primers to use. I tested  these 2 primers
> set. I got a signal for first three PCR reactions, but after  (e.g. other
> PCR reactions with the same cDNA) the PCR signal (band intensity) decreased
> until it disappears.
> I diluted the primers in sterile water. The primers were used at 0.25uM,
> dNTP at 5mM, TAQ (Perkin Elmer) 0.75 units and with Perkin Elmer buffer.
> Total volume : 25 uL. The PCR reaction, RTases and electrophoresis on
> agarose are good since our positive control (human) are good.
> Can we dilute primers in some buffer instead of water. If yes, which buffer
> ?
> Is there a special task with porcine primers?
> Does water have any effect on the primers?
> Any suggestions ?
>
> Thanks a lot !






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