5 replies to this topic

[vgupta]

I have been contamination problems in my no DNA control of PCR. I have changed all the solutions and have got new primers made. But some how this contaminating band always appears. This happens with a particular DNA clone and not with other clones. Also there is one particular seuence of primers which doesn't give me contamination with this clone. I set up the reactions in a hood. I don't know what to do?

Edited by vgupta, 30 June 2004 - 12:37 PM.

[phdconsult]

Two sources of contamination that produce a band in the No-DNA control are:

1. Mycoplasma contamination with a genome GC strength of 27.5%- it is everywhere; including the HEPA filter of your hood.

2. Your primers could carry linear adducts of the molecule resulting in lengths upto 250 nucleotides. This happens due to condensation reactions that were not arrested at the right time during thesynthesis. If you ordered new primers but did not change the manufacturing company, maybe you should. Run a sephacryl S-300 gel filtration on your primer batch and check for more than one 260 nm absorbing fraction.

[vgupta]

Thanks for your suggestions. Do you think primer purification or mycoplasma contamination could be a problem even if the band is of the same size as the product expected in positive control (with DNA)? I am trying to amplify a prokaryotic vector sequence integrated into mouse genome. My primers are from qiagen. One particular set of primers works well with this construct.

[ocean]

vgupta, on Jul 1 2004, 07:57 AM, said:

Thanks for your suggestions. Do you think primer purification or mycoplasma contamination could be a problem even if the band is of the same size as the product expected in positive control (with DNA)? I am trying to amplify a prokaryotic vector sequence integrated into mouse genome. My primers are from qiagen. One particular set of primers works well with this construct.

have your solved your problem yet...i group member had the same problem and it worked out that the gloves was the source of contamination

[perseo]

Hi, I have the same problem, the negative control ( without DNA template) presents bands of the same weight as the expected ones. It's rare because in poliacrilamide gel the band in the negative control have the same pattern of the other bands showing even heterocigocity. I dont have a positive control because I'm trying to tranfers primers from another genus. If there was a contamination of a strange DNA why the band show Microsatellite patterns?

The only option I can imagine is that somehow the DNA of my samples are contaminating my PCRs. I Dont know haw because I worked in clean conditions.

I need some advice. Thank you

[klinmed]

vgupta, on Jun 30 2004, 09:30 PM, said:

I have been contamination problems in my no DNA control of PCR. I have changed all the solutions and have got new primers made. But some how this contaminating band always appears. This happens with a particular DNA clone and not with other clones. Also there is one particular seuence of primers which doesn't give me contamination with this clone. I set up the reactions in a hood. I don't know what to do?

It is almost impossible to remove all DNA contamination from polymerase preparations. Hence all batches of taq contain low levels of prokaryotic/phage sequences (from E. coli in recombinant taq). This can be a major problem and may be the cause of problems with your primer set.

To see many papers on this subject Google "taq bacterial sequences".
You may be able to overcome the problem by changing primers or switching to natural (T. aquaticus) taq.

For example: Spangler R, Goddard NL, Thaler DS, 2009 Optimizing Taq Polymerase Concentration for Improved Signal-to-Noise in the Broad Range Detection of Low Abundance Bacteria. PLoS ONE 4(9): e7010. doi:10.1371/journal.pone.0007010