I am trying to amplify part of the 16S bacterial gene. I would really appreciate some advice. Running the PCR product on the gel, I obtained one specific band at the desired size and another specific one at about 1500 bp. I have tested all the options that may affect i.e. increasing temp, decreasing enzyme concentration, decreasing annealing time, decreasing extension time, lowering the number of cycles, but no success.
Does anyone have any ideas?
What is the Template? Is it from a colony or genomic DNA or RNA prep?? What is your expected size? What were your controls?
What is concentration of MgCl2 in your PCR setup, it should't be greater then 1.5mM
@Vingesh, it is gDNA. Expected size is 740 bp.
@Dinesh 2 mM MgCl2. But it did work very well with other DNA samples.
@Mohamed Yes I did, no success.
@ Nadine This is a special primer from the sequencing centre and I can not change the primer.
I agree with Nadine. Cut it out of a gel and use it for your purpose. You can also re-amplify it after cutting and purification to have good amount for subsequent analyses.
Even if you will decrease the amplification of non-specific band and it will not appear on your gel, you will get some amount of unspecific product, that will disrupt your sequence later.
As mentioned earlier, I would also cut the band out of the gel and send for sequencing. I don't know the sequence of primers, but they may be complimentary to multiple regions on your DNA fragment, so you might be getting multiple products.
Dear Mahdi
Plz accept this book as a gift from me. it will help you a lot for PCR and downstream procedures.
Best Regards
just a general comment after I saw the gel picture now, you only ran the gel half the length because the markers still got a whole lot to run and be separate from each other. The bands look pretty ok and if its the correct size, just send them for sequencing. The bands at the lower end of gel might only be the primes or primer dimers.
If the strong band in your picture is the correct one, then this looks like a typical PCR product pattern, with some weak "primer dimer" near the bottom of the gel and some other faint bands at the top. You could probably get a cleaner pattern by diluting the DNA template at least 1/100 or even 1/1000. Remember that there are probably multiple copies of the rRNA gene in your bacterial DNA and the effective concentration is more than 1000x higher than for a mammalian DNA, where 1 ng of template and 30 cycles is quite enough to get a respectable product from a single copy gene.
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