Help... my PCR don't come any more - PCR amplification desapeard (Aug/18/2010 )

Hello everyone… first of all, my English is not so good, so I hope you understand me…

Well I’m working with this gene from 2 and a half years and I have had lot of problems with it…I do PRC and Sequencing of it to search point mutations. I work with DNA from micro dissected paraffin tissue samples.

I can not understand why the PCR comes and 2 days later it doesn’t. I have 3 pair of primers for the same region; one amplifies at 118bp, the other at 225 and the third at 257bp. They were all coming great but now I have no amplification at all…

I’ve performed a series of tests with different TM 56-60 and different MgCl2 concentration too 0.5-2.5mM and I could amplify something with the 257bp primer. I did the purification and sequence. They came nicely…

Then I tried again with another group of samples (I always use a Control sample), made an electrophoresis and I great bands again… So, purification sequence and then THE HORROR AGAIN…When I tried to read the results I got a sequence that didn’t match my gene, it was a complete different sequence…

The other 2 primers are still not coming. I’ve changed everything, Taq, Buffer, MgCl2, dNTP’s, Primers, and water… Please let me know if you have any idea!!!!!!!

-GJKoup-

Sample degradation? make sure you don't freeze/thaw your samples too much.

-bob1-

I'm not sure what is happening... It doesn't come neither with the new ones. I’m thinking it could be the extraction. I’ve change my extraction kit. First I was using the Quiamp FFPT DNA extraction kit (Quiagen) and now I’m using Pure Link Genomic DNA Kit (Invitrogen). Some samples come and some doesn’t. I’ll try using more PK at the digestion point.

What about the missmatch sequence that I'm gettin???

-GJKoup-