I managed to extract DNA from two marine Actinobacteria strains only after using PowerSoil DNA Isolation kit (MO BIO) with heavy load of colony sample. Sample collected from Petri dish. [The UltraClean Microbial DNA isolation kit (MO BIO) using small amount of sample did not work (no band seen on gel), even after previuos repeated freeze-thawing or heating of sample at 70ºC for 10 min.]. PowerSopil gel extraction showed one neat band of gDNA for each sample. However, I can't get primers 27F and 1492R to amplify fragments from the gDNA. Have already tested various proportions between Taq, MgCl2 and dNTPs, all resulted in very poor or negative results. Tryed adding BSA with no result. In all cases the positive controll amplified and negative controll was clear.
What could be wrong? Heard DMSO could help. Any ideas?
Up-to 5% final concentration of DMSO with the addition of BSA is definitely an option in case of secondary structures, just make sure your buffer don't already contains DMSO.
I would also try a touchdown PCR protocol in-which you start with 65c initial annealing temp and drop it by a deg for the first 10 cycles, then continue with additional 20 cycles at 55c. Alternatively, you could do a gradient pcr to test for the optimal annealing temp for your product.
Another option is to try and see if you could get amplification of a smaller product say just the V3, this would test the quality of your template.
I think a lot of times it's the taq. I've failed gradient PCRs using many variations using Fisher-taq and then Platinum-taq gives amplification no problem. If I remember correctly, Platinum comes with it's own buffer optimised for that taq. I would encourage you to try a higher quality taq.
For the DNA extraction, did you bead beat or just heat to 70C for 10min? Just heating for 10min sound like a pore way to lyse a gram positive bacterium. Freeze-thawing is also quite a weak lysis technique. With a proper bead beater you can gently lyse them or absolutely destroy them, depending on your settings. Also, powersoil kits tend to have really low DNA yields but good purity. It's a tradeoff on purity vs yield. They do so many cleanup steps you'll lose a lot of your DNA vs maybe MP Bio FastDNA Spin kit or Ultraclean with a bead beating step. For your samples, you probably don't need all the cleanup steps.
Dear Claudia
You haven’t mentioned about the concentration of gDNA? Have you quantified your gDNA?
The right/optimum concentration of gDNA is also very important for performing PCR. Too high or too low concentration may be one of the probable cause of your PCR failure.
Hello, Ido, Steven and Sunil. Thanks a lot for all suggestions given.
Steven, the refered MoBio kit uses bead beating. We heated samples at 70ºC/10min before extracting with the kit. No or poor results. With PowerSoil, we used heavy load of colony sample. Nice band on gel.
Sunil, I have no idea of DNA concentration, but can assure it is high due to intensity of the band compared to the positive control of known concentration. We tried amplification with concentration as is and diluted to 10%. No results.
Ido, we will try DMSO+BSA.
We will also try another extraction using lysis with lisozyme, followed by PureLink gDNA mini kit (Invitrogen).
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