PCR 'contamination' - (May/14/2007 )
Hello guys it almost 3 week contamination is giving me headache. Each time I run PCR I'll see something/ "Band" brighter and thicker but smaller (<100bp) than positive products. I tried to change the annealing temp but it did not work. This 'band' is not consistent some times it appears in all lanes some times in negative controls....so confusing! pls help!
It really seems a contamination 'cause you find it also in negative ctrl, surely you tried changing reagents, using lower annealing temp....so are you sure of specificity of your primers? have you tried changing aliquot or to re-order them?
may be also the environment (some plasmide in the air?) or pipettes not very clean or not used only for PCR by error.
hope this could help you.
Good Luck!
Are you using filter tips?
Hi I am not using such kind of tips. I am using the ordinary tips after autoclaving and of course only for PCR. But i accept 'may be also the environment (some plasmid in the air?)' by Zaybo....ya I am working on bacteriology bench...I'll try in separate room...and inform you.
Thank you!
did you check that your water is sterile?
Hi I am not using such kind of tips. I am using the ordinary tips after autoclaving and of course only for PCR. But i accept 'may be also the environment (some plasmid in the air?)' by Zaybo....ya I am working on bacteriology bench...I'll try in separate room...and inform you.
Thank you!
I agree with Dayo, if you use filter tips you really should be ok, I know they are more expensive but they are definately worth it.
You really should use filter tips. In fact, consider them a necessity for PCR. Once, I ran out of filter tips and started using normal tips for PCR - for the first time ever - and it was fine! No contamination! So I thought they were over-rated, an unnecessary expense... then, after a few weeks, contamination started to appear... and once you get it it's hard to get rid of it! Changing all the reagents doesn't help!
So that's why I consider the #1 cause of PCR contamination to be dirty pipettes. Particularly p10 pipettes. In a perfect world, all tips would be filter tips...
The size and variable appearance is typical of primer dimer problems.
If you do not want to quantify your product, than this primer dimer (I second tfitzwater in his opinion) should not be much of a problem. Some garbage below 100bp can be found in many PCRs. And, I had it once, that the company mistakingly modified the primer by adding FAM - this made very bright spots at the end of the lane...
i think it wld be of great value if i cld post the gel photo but I failed wld u teach me how...so it will be very clear to everyone what I mean by 'band'...
10Q