16S rDNA PCR protocol- please help

[Alkendi]

hi all

I need your help in this matter....I am stuck I guess!!!!

I collected manure samples from the following animals:cow, deer, pig, chicken, chicken litter. Then I amplified the 16S rDNA from each animal DNA using universal primers 515F/1492R. I could amplify the 16S rDNA gene from all of the animal samples except the deer. The PCR protocol I am using is:

94C for 10min

35 cycles of the following 3 steps:

94C for 1min

50C for 1min

72C for 1.5min

72C for 10min

4C for hold

the previous protocol worked for all animal samples except the deer sample!!!

I used Fast Prep kit to extract the DNA.

The melting temperature for 515F primer is 63.8 and for 1492R is 49.4 and as mentioned above I am using an annealing temp. of 50C . I tried to raise the temp. to 63C but I still don't see any bands. I usually load 13.4ul of the DNA sample to the master mix (total of 25ul).

If you can help me figure this out please reply .

[Paralith]

13.4 ul of DNA? that's a lot of DNA, my friend. Unless your samples are at a VERY low concentration, you shouldn't need that much. I typically use 1 uL of DNA sample (which is usually at a concentration between 80 and 200 ng/ul) in my PCRs. Using too much DNA can actually prevent the PCR from working correctly, so I would use less template DNA if I were you. It's also possible that the primers aren't very specific for deer 16s, though I think that's unlikely. If all else fails try lowering the melting temperature.

[Alkendi]

I use 13.4ul of the DNA but it is 10^-5 dillution. the other thing is that this procedure worked for the bacterial DNA extracted from the other animals but not for the deer!!!

[Paralith]

well like i said, maybe the primers aren't very specific and don't match up with the sequences from the deer sample very well. Try lowering the melting temp if you don't think the DNA is the problem.

[CharonY]

The protocol appears to be find. Also, the primers should not be a problem (they are pretty standard).

Increasing the annealing temperature will lower the yield (but increase specificity). The following things should be checked:

- was the DNA extraction from deer manure successful? Check the DNA quality and quantitiy.

-is the DNA pure enough? Contaminations can ruin the PCR esp. if you use 13 µl of template

-are the components still OK? Make another PCR with a positive control to check that