unknown PCR bands - (Jan/24/2006 )

Hi
I'm doing PCR on a metabolic gene, and every time I check PCR products on a gel, I get discrete bands of 100 and 75 bp, and sometimes 200bp. My product is supposed to be 150-200 bp, but I don't know whether this gene is present in my sample.
Another issue, is that I get same bands in my negative(water only) control!

I did PCR several times with recommended cycles, and once I didn't have anything in my 0-control, but now I have it again!

I also checked the primers with BLAST, and most hits are from the right gene, but the rest are from human, mouse and some other bugs, and our labs doesn't work with such stuff. (unless it was me sneezing?)

Can anyone help me with that please??

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Unfortunately, if you are getting bands in your negative control then it is more than likely that you have a contamination in your reagents/DNA/water.

I would suggest using new aliquots of all of these, and atleast getting rid of the contamination first before you can tackle your PCR band problem.

Also, check the self complementarity of your primers, often you will get "primer-dimers" at about 50-75 base pairs...

good luck

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I also did 16S-PCR with the same reagents (except primers) and same samples, and didn't get anything! My positive control used to work, but now even +cntrl does not!

This doesn't look like primer-dimers, since these are bands, and not smears. I don't know if primer-dimers look like bands, at least I've only seen them as smears.

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Here's a trouble shooting guide to "i got bands that shouldn't be there":
for smaller bands:
Increase annealing temperature
Increase annealing time
Increase extension time
Increase extension temperature to 74-78º C
Decrease KCl (buffer) concentration to 0.7-0.8x, but keep MgCl2 concentration at 1.5-2mM
Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant
Take less primer
Take less DNA template
Take less Taq polymerase
If none of the above works: check the primer for repetitive sequences (BLAST align the sequence with the databases) and change the primer(s)
Combine some/all of the above

for bigger bands that shouldn't be there:
Decrease annealing time
Increase annealing temperature
Decrease extension time
Decrease extension temperature to 62-68º C
Increase KCl (buffer) concentration to 1.2x-2x, but keep MgCl2 concentration at 1.5-2mM.
Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant.
Take less primer
Take less DNA template
Take less Taq polymerase
If none of the above works: check the primer for repetitive sequences (BLAST align the sequence with the databases) and change the primer(s)
Combine some/all of the above

if you get bands in a "water only" control, you got a contamination. Change water, and other ingredients. Wash pipettes, or use different pipettes for PCR only.... if you look around in here, you get to know the drill.

Vetticus

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Thanks Vetticus!

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