Trouble 1: I ran a PCR reaction and found a low molecular weight band (100 bp) when doing electrophoresis. 1ng plasmid (OD260/280=1.84) was used as template. Final concentration of primers was 0.2uM. I thought that band might be primer dimer. So I ran primer solutions on a gel again without PCR, but i can still see the band. Everything was newly prepared. Length of each primer is 20bp.
Trouble 2: ddH2O was used as negative control but an obvious band (with my target size-1100bp) can be seen. So I ran water directly without PCR and there was nothing on the gel. I freshly prepared water and did PCR again and the 1100bp band appeared again.
How could the above happen? What should I do?
I also think that the small band is primer dimer. The annealing temp for about 6 bases to anneal will be less than room temperature and the taq is still active at low temperatures. You can use a hot start enzyme or set up on ice and transfer the tube onto a preheated pcr block then immediately start cycling.
2 When the water amplifies that well you have post pcr contamination in a reagent or on the plasticwae, bencharea or pipettes.
Thake fresh primer aliquots and move to another researchers working area and use their pipettes to set up a pcr with 3 no template contrils (water) and one positive contril sample.
Meanwhile clean your pipettes by stripping them down and wash with soapy water. Clean your working area with soapy water. If the pcr works well then introduce your reagents and see if the contan=mination appears. if not then move back to your area and set up the same pcr to see if the contamination is in your working area especially if you run checker gels in your area
Thank you, Paul. I will try your advice. My pcr protocol is 95°C,3min - 95°C, 30sec - 55°C,30sec - 72°C, 30sec (40cycles)--72°C, 2min - 4°C. Tm of primers is 60°C. Do you think i need to change the parameters? Another question is for the electrophoresis result of without PCR (last band in the left image) , the primer sample should give no bands right? Why can i still see the bands at same position as the post pcr one? Paul Rutland
Trouble 1: It is not obvious from your picture that this is not a dimer. Electrophoresis should be carried out longer.
Trouble 2: I think you have contaminations in your water. Old water contains more DNA, which additionally gives a band of about 250 bp. In new water there is much less of this band. However, it appears that there is more DNA that produces your specific band. Both waters are contaminated. To avoid such problems, buy commercial water for PCR, or you can even use water for injection from a pharmacy.
Hello Anita,
the small blurred band without pcr is primer or primer dimer. Even single stranded dna will self anneal and form loops and the double stranded sections will bind more EtBr and allow you to see either a single primer or both primers and the more annealing there is the stronger the fluorescence.
I would try extension of 40seconds to improve the efficiency of the amplification and look carefully at the amont of dna template you are using. 40 cycles is a lot and I would expect good amplificaion at 35 cycles or fewer so it may be that you are using too much template and getting pcr inhibition of the pcr so using less template (or better quality template) woud work better
When you get amplification of water to give a long pcr product in the negative control thenyou have contamination of plasticware or a reagent or in your working area or pipettes which can be dealt with as in my first answer
The band in your water is contamination. You need to throw away ALL (and I do mean ALL) of your reagents (enzyme, buffer, water, primers, dNTPs, etc.). Make a new batch of TE buffer & make a fresh aliquot of your primers.
Get new boxes of pipet tips & other plasticware. Wipe down your bench area & clean your micropipettes. Open a new box of gloves.
Common sources of contamination:
- cross-contaminated solutions (e.g. your tube of water)
- open-top disposal containers for waste tips
- dirty soaker paper or other items on your bench
- your micropipettes, especially if you use the same ones for handling amplified DNA samples (PCR products) and setting up reactions. This is a BIG mistake. Your lab should have a separate micropipette specifically dedicated for loading PCR samples into agarose gels.
Good luck!
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