5 replies to this topic

[Sherina Kamal]

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.

[phage434]

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).

[Annalucinda]

Please ensure that your primer is working efficiently now.

[Sherina Kamal]

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

[phage434]

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.

[Sherina Kamal]

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