PCR bands in NTC Control and Neg Control Lanes - (Mar/02/2009 )
I am currently trying to amplify gDNA to determine if a gene of interest is in my human cells. I have isolated the gDNA and I am looking for some help in figuring out why I am getting PCR bands in both my NTC control lanes as well as my Negative control lanes. I have included a positive control in the reaction which amplified beautifully, however I am getting a band at the EXACT same size in my NTC and Neg lanes. I had originally though that I had contaminated primers, but I re-ordered them and I still see the band. I have used all filter tips for setting up the PCR and I have used a new Taq MasterMix kit for each run I have completed. The water in the samples (and the water used to resuspend and dilute the primers) is UV irradiated RNase free water. If anyone has any ideas for me please let me know. I am working in a lab where no one does PCR and there is no one here who is able to help me at all. Thanks!
Human cells and DNA are everywhere. How many cycles are you doing? With enough cycles, you could probably amplify a human gene from almost anything. Think about everything that comes close to the tube. Do you autoclave tips or tubes? Perhaps the autoclave is contaminating things. UV treatment of water won't solve contamination problems. Set up and store your reagents away from the cycler, and especially away from any amplified DNA products. Ideal would be a different lab or different bench. Are you certain you have a product, and not primer-dimers?
Thanks for responding. I am currently doing 35 cycles for the PCR. It was what was recommended by the manufacturer of the Taq. We autoclave everything that I use for set up, except the PCR tube itself. If you do not autoclave how can you be sure the materials are sterile? I set up and store all of my reagents at my own personal bench, which is on a different lab bench than our thermocycler. I'm not sure how I can control for amplified DNA, as my lab works with many different DNAs, both plasmid and human, on a daily basis. I am certain the bands are not primer-dimers. I can see the primer dimers on the gel, running a little below 100bp. The band I am looking for runs at ~230bp, and this is the band that I am seeing in both the positive and negative control lanes, as well as in my samples of interest.
jdklove18 on Mar 2 2009, 01:34 PM said:
It does sound like you have contamination issues. If you order all your supplies already sterile, you don't need to autoclave them. 35 cycles is kind of a lot, even if you have only a very small amount of contamination, 35 cycles will amplify it - maybe try to drop down to 30 cycles if you have a good strong band in your positives. Good luck.
Sterile is not clean. I would use tips and tubes directly from the box. You don't need sterility for a PCR reaction. If you need them sterile, buy them sterile, but this is unlikely to be necessary. I would run some positive samples and negative samples and run gels at 25, 28, 31, 34 cycles, or (better) run samples on a QPCR machine to see when the NTC shows amplification. I'll bet you are amplifying very weak samples.