I look for variation in strain bacterium and I want to do sequencing
but I have a band in negative control that same in my strain bacterium
first time I noticed band in my negative control, I checked my primers and master mix that without contamination but again I had a band in negative control again what is the reason?
The short answer is that you have contamination. Throw away ALL of your reagents (buffer, enzymes, primers, water, etc.), clean your work space, don't use the same micropipettes for setting up PCR that you use for handling amplified PCR products, and try again. Good luck!
as well as Katie's advice clean your working area thoroughly and strip down your pipettes and clean them before using them again. I agree that you do have contamination and even tiny amounts of amplified product on gloves or in spilt electrophoresis buffers or in the dust in the lab will amplify well. Every 10 cycles of pcr is 1000 times more product so you do not need very much contamination to produce a visible product
dear paul rutland
thanks your answering ...but i did your suggestion..but i had band ....i and my friends have this problem...even i try reagents of my friends...but apear band
what is your idea about impurity tag-polymerase in master mix?
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dear katie A burnette
thanks your answering what is your opinion about impurity tag-polymerase in master mix?
there's problem in your primer that you choose it therefore the negative control of strain bacterium showed the primer (mean the primer it's actually in negative control)
It's possible to contaminate any of your reagents, including the taq enzyme. That's why I'd recommend you throw away all your PCR reagents, even the enzyme. It's not worth tracking down exactly what was contaminated as long as you can get rid of it.
dear shaima R.banoon
thanks your answer...size of my band is 914 bp in my negative control
in your opinion this band is related to primers?
Dear Shadi,
any reagent or reagents could be contaminated and you have 2 options. First throw everything away and have a major clean up of all equipment,working areas etc.
Second design a new primer outside of the primer set that you are now using and then the contamination cannot amplify. This will work but if your working practice is not excellent then eventually you will get contamination of the new primer set amplimer.
concerning your question about the Taq. Many years ago when people grew their own taq and purification was rudimentary there was a problem of taq dna in the enzyme which could rarely anneal to some primers and give a product but I think that this is not the case with your taq because you said that your pcr used to be clean but is now amplifying a band from the negative control , If the taq was naturally impure then the problem would have been with you from the start. It is characteristic of post pcr contamination that it only occurs after some successful pcrs
dear dominiki heppele
thanks your answering..but all of my staff was autoclaved...and i was be very careful..
Dominik Hepperle
This is my recommendation: Do NOT use autoclaved pipette tips and Eppendorf vials. You may get a contamination while autocalving because the organisms might/should be destroyed but not their DNA.
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