PCR Contamination issues - PCR false positives (Oct/01/2004 )
I use to have the same problem but one day it stopped. I still donīt know why but I really donīt want to know
someone mentioned UV. irradiation of pippetes and microcentrifuge tubes. This makes good sense to me because in my case i suspect them for my PCR contamination issues. How do you actually go about it. do you use the UV of the laminar flow? because that would create a panic in my entire lab. people in my lab are scared of UV rays! so where should i expose my tubes and pippete tips to uv and for how long?
Use the UV of a closed laminar flow (good flows won't allow UV to turn on if the flow isn't closed!) and put a paper stating when you will stop the UV.
I have fluoresence in my water control which is not due to contamination. Some papers are actually stating that they are getting this, some call it autohydrolysis of the probe, however when the fluoresence is as reproducible as mine >30 high normalised fluoresence, it has to be due to something else, some chemical in the water I presume. I used DMPC-treated water and RNase free water from different companies, I even used water from the tap, the fluoresence was exactly the same. Until I used PCR grade water which gave a much reduced fluoresence and also later Ct values. I suggest using PCR grade water and water for injection to dissolve primers.
HI!
Please all help me out as i am doing eNOS gene amplification of 457bp I got the band when i did PCR .But now days i am not getting any thing just a streaking.Please help me as i am very upset......... I am using annealing temp 56.....
Hey guys,
I have been struggling with a contamination problem for 2 weeks now. I tried changing the water, the reagents and the primers but I still have bands in my negative control. When my lab mate tried with my reagents and her primers, the bands did not show up. I tried with her primers and my new primers but they still appeared in my negative control on my gel. I do not see any difference in our techniques, or the way we make our PCR master mix. The only thing I have not tried until then was changing my PCR tubes. I autoclaved my new PCR tubes and we both tried doing PCR with same the reagents and primers. Negative control bands did not show up. We thought our problem was solved but after two days they started appearing in my and her negative control samples. So now we both have the problem. The weird thing is they are not same for all my runs. They are changing from one run to another run. Sometimes I can see only one band that is further from my 100bp marker and after the dye mark (I guess it is a primer band) and sometimes there are multiple bands with my primer band. From these results, I can say there is something affecting my PCRs in an off and on fashion. After reading all your suggestions from this forum, I have cleaned all my stuff with bleach and tried PCR. Still there were bands in my negative control. The only thing I did not try was cleaning the internal tubes of my pipetters. I am planning to try that tomorrow. In the meantime, if I can get any suggestions from you, that would be really helpful for me.
Thank you all !!
1) barrier tips
2) don't autoclave tubes or tips or anything else that comes near a PCR machine
2) don't autoclave tubes or tips or anything else that comes near a PCR machine
Thanks for suggestions!!
So are you saying tips can be the cause?
If I do not autoclave tubes and tips how can I maintain them sterile because I don't buy tips in boxes.I buy them in packets and I will autoclave them.Regarding tubes,I will buy them from VWR,I do not know if they are sterile or not .So I will usually autoclave all the stuff I use in PCR.
I just did PCR for my water in 3 tubes.I did not see any bands in it.So from this result,can I conclude my tubes,pipettes,tips and water are not contaminated?
thank you.
Barrier tips prevent contamination of your reagents by accidentally overpipetting into the barrel of the pipet. No PCR reaction needs to be sterile. Tips that come out of the bag or box are clean, which is much more important than sterile for PCR. Touch tips and tubes with clean gloves (a new bag if others are handling them). Have you ever looked at the water in your autoclave?
Today I figured out the bands I am seeing are not because of the contamination.They are because of the primers-dimers.But When I used same set of primers one month back that band did not show up.What ever the primer set I do use now,primer-dimer band is showing up.I do not know what is the reason for that.Can you guys suggest me what could be the reason for this.
Thank you!!