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16S rDNA PCR protocol- please help


Alkendi

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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 .:confused:

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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.

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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

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