This week I did a colony PCR of three bacteria I want to identify (16S rRNA sequencing). I did this by touching a colony with a sterile pipette tip and resuspending it in sterile water (in triplicate). I used E.coli for the positive control and sterile water for the negative control. Then heated at 95°C for 5 minutes and centrifuged at 13300 rpm for 1 minute. I then added 2 µL of the supernatant to 23 µL PCR master mix. I used taq polymerase and primers 27F and 1492R.
I used the following PCR program with 30 cycles:
95°C 10 minutes
94°C 30 seconds
46.5°C 30 seconds
72°C 1.5 minutes
72°C 10 minutes
Then I performed an agarose gel electrophoresis of the PCR products. The positive control shows bands and the negative control has no bands, so the controls are correct. Bands are also visible in one bacterium, but nothing is visible in the other two bacteria.
Does anyone have any tips for me so that I can see bands for the other two bacteria that I want to identify?
As both of your controls worked as they should it may simply be a case of the positive control having more dna template than the colony. You could try a pcr with different tubes having 2,4,6,10 ul of your dna
I have also had situations where I had added too much of the DNA template from a colony extraction. In addition to going up in DNA concentration, I would try a few with less (such as 1 and 3ul of a 10-fold dilution). You can sometimes get inhibitors from all the cell debris.
I think the annealing temperature is low in this case. In my opinion, you can use 54 degrees instead of 46.
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