Originally Posted by Alicialsw
Argh!!! I'm having a pretty bad week as I've been getting bands on my negative controls. I've done negative controls without template, without primers and without dNTP. I've even change the water every single time. The problem I'm facing is, I'm getting the same exact size band with my expected size and to make things worse, the band is even brighter!!! I've tried using my colleagues pipette, water, PCR reagents and rediluted my primers from the stock primer solution but nothing seems to be working. I kept on getting bands on my negative control (minus template) which is the exact same size with my expected band (which I sequence and is the gene of interest).
Please help me out here. I'm trying my very best to troubleshoot but I have no idea what is the contaminant. Sigh......
There must be alot of contamination floating around if no dNTPs/no Primer neg controls still show a band. Meaning primers and dNTPs are also available from whatever contaminant source.
1st. Are you new to doing PCR? If so you need to work on your technique, etc etc. [Don't want to bore you/discourage you by harping on this; only you will know if it applies to you]
2nd. You need to clean up, wipe down your work area, and any area nearby. Use Ethanol/Isopropanol or Bleach.
3rd. Toss reagents. Get new reagents (even stock primers), autoclave water, tubes, tips, the whole works. Take your pipettors apart (carefully) and decontaminate them (ethanol or 10% bleach). Use UV lights for decontamination at the end of every single day, if available.
4th. Separate the extraction, PCR setup and PCR machine and gel electrophoresis equipment out. Run them in separate parts of the lab or even rooms if available. (this is to cut down on aerosol contamination).
5th. Use gloves and change them frequently.
6th. Don't store reagents in same fridge/freezer as any pcr amplicons/gDNA.
Keep them separate to avoid contamination, don't want them in the air when you open a reagent or PCR rxn tube, or landing on surfaces that will be touched and the contamination carried into new PCR rxns.