Didint get any band during gel electrophoresis even though DNA was there - (Sep/20/2011 )
Ive extracted my DNA previously with general bacteria primers (357 F(GC) and 518R) with two different concentration of DNA 2 and 5 microlit.
Now im trying again with the same primer, same protocol, its not working. No band appear and i wonder why. Ladder was nice separated. Scared that the DNA was a lot. Ive also tried to dilute the DNA and check with the purity, its 1.88 260/280. But still, when I run the PCR with the same primer and protocols, no band appeared.
PCR programme: 95C-5min; 10cycles of 94-30sec,55C-30sec,72C-1min; 26cycles of 92C-30sec, 52C-30sec, 72C-1min; 72C-10min
I still have long way to go as I need to get the list of sulphate reducing microorganisms but the general bacteria itself is hardly to get.
What enzyme are you using? The denaturing temperature could be too low (especially in the later cycles). I would try 95 for all of the denaturing steps (unless using Phusion, when it should be 98).
Please ensure that your primer is working efficiently now.
meaning that we should not follow exactly what the literature review state? im referring to paper written by dar et al., 2005. so i should try the denaturing step at 95C?
im not sure the enzyme, will let u know tomorrow.tq
Yes, that is exactly what I mean. There are many protocol errors in the literature, and your equipment differs from theirs. Use your own judgment, always, especially if you are having problems.
Ive tried to dilute my DNA and I used that to run PCR and finally I got few bands yesterday. but it wasnt a very bright one, so i need to increase the number of diluted DNA to be mixed with the primers, mastermix and nuclease free water