Edited by vgupta, 30 June 2004 - 12:37 PM.
PCR contamination in negative control
Posted 30 June 2004 - 12:30 PM
Posted 30 June 2004 - 02:27 PM
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.
Posted 01 July 2004 - 06:57 AM
Posted 21 September 2004 - 02:11 AM
vgupta, on Jul 1 2004, 07:57 AM, said:
Posted 03 December 2009 - 11:57 AM
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
Posted 03 December 2009 - 01:20 PM
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