Help... my PCR don't come any more
Posted 18 August 2010 - 10:50 AM
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!!!!!!!
Posted 18 August 2010 - 01:01 PM
Posted 19 August 2010 - 10:11 AM
What about the missmatch sequence that I'm gettin???
Posted 19 August 2010 - 10:12 AM
What about the missmatch sequence that I'm getting???