RAPD PCR breakdown

Dr. Klaus Eimert eimert at web.de
Thu Mar 25 05:21:28 EST 2004


amyezers at kings.edu (DrAnnie) wrote in 
news:9c64828.0403231006.5af557ee at posting.google.com:

> Some info that might help:
> 
> 1)  All of my primers are from Operon and are 10 mers.
> 
> 2)  Some are older than others but even the fresh ones aren't working.
> 
> 3)  I have over 100 primers, so I'm not going to list the sequences.
> 
> 4)  I've used the same MJ Research PTC-200 this whole time. 
> 
> 5)  After an intial 4 cycles that include a ramp down to a 37 degree
> anneal, then there are typical 35 cycles with 94 degree melt, 37
> degree anneal, and 72 degree extend with an additional 10 minute
> extend at the end after all of the cycles are complete.
> 
> 6)  Used the same 96 well plates and seals.  Tried to use the same
> reagents, but companies have been sold and bought since I started
> (including Operon) and so the reagents come with a different name, but
> I am assuming the contents are the same as listed.
> 
> 7)  Have tried fresh dNTP's, Taq, templates, etc. to no avail.
> 
> 8)  Am about to try a run with snap freezing the primers.
> 
> Is there anything else I could tell you that would help?

Hi,
it may sound silly - but did you check whether your water is still OK? 
Had this happen to me once ...
Cheers,
Klaus



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