Cre genotyping - smeared results (Jul/21/2008 )
Hello, I was wondering if anyone could help me troubleshoot my Cre PCR. I have been getting smears that wash out the results for quite some time now. I have completed the following to fix the situation, and none of it has worked!
- changed all products and optimized sterility
- diluted DNA to proper ng conc.
- washed DNA to clean off possible remaining alcohol from isolation
- double checked primer conc.
- repetition
- water controls - brand new water bottle showed the smear
- ordered new primers - same smear
any ideas? What is everyone using for the annealing temp for Cre PRC. I am at 58 degrees. I am going to try 60 or 61 next.
the DNA is isolated using HotStart method (NaOH at 95 degrees then neutralize), also have done overnight is lysis buffer with isoprop wash.
- changed all products and optimized sterility
- diluted DNA to proper ng conc.
- washed DNA to clean off possible remaining alcohol from isolation
- double checked primer conc.
- repetition
- water controls - brand new water bottle showed the smear
- ordered new primers - same smear
any ideas? What is everyone using for the annealing temp for Cre PRC. I am at 58 degrees. I am going to try 60 or 61 next.
the DNA is isolated using HotStart method (NaOH at 95 degrees then neutralize), also have done overnight is lysis buffer with isoprop wash.
I figured it out I was using a 1.5mM [MgCl] and a 58 degree annealing temperature which our lab had been using for over a year. I ran a cascade PCR with different annealing temperatures and 4 [MgCl]'s and it turns out that anywhere between 43 and 52.2 degrees annealing temp works, and for more specific biding use 65.5 degrees. The more pivotal aspect is the MgCl which needs to be used at 1mM.
Thanks for the help BioForum, I mean, myself, because no one responded
