PCR help needed
Posted 24 August 2009 - 01:20 PM
I have gotten the primers to work on a closely-related species for the same fragment but can not figure out whats going on. Im working with mitochondrial DNA and the absorbance numbers are pretty good so I dont think it is the template. I attempted a few different concentrations of DNA at 54 degrees and they did not work.
Can somebody give me any suggestions that may push me towards the right direction? Im pretty new to the wonderful world of molecular technology so Im a bit confused on what to try next.
Posted 24 August 2009 - 01:53 PM
I'm guessing that you're isolating DNA from bacteria? Try adding adding a little DMSO to your reaction--here are the conditions I used for a 16S rDNA product:
H2O: 28.45 ul
Buff: 5.0 ul
MgCl: 4.0 ul
DMSO: 2.5 ul
Primers: 1.25 ul ea
dNTP: 1.0 ul
Taq: 0.3 ul
gDNA: 6.25 ul
Edited by lab rat, 24 August 2009 - 01:54 PM.
Posted 24 August 2009 - 02:17 PM
I will still consider the DMSO, thank you!
Posted 24 August 2009 - 02:30 PM