Previously I genotyped a 96-well plate containing genomic DNA from cell lysate. I was able to get a band at the desired location, however I am trying to reproduce the genotyping results for a single colony that was positive and cannot get the gene to amplify (no band).
Using Onetaq hot start mastermix ploymerase mix, I tried a annealing temp gradient in which I was able to get bands in every temp but now when I try the same annealing temps, I get no bands even though this specific colony produced a band when I genotyped the entire plate.
If anyone has any trouble-shooting suggestions on how to fix this issue it would be greatly appreciated!
Thanks
Does your pcr experiment include a positive control sample that will always amplify to show that the pcr conditions and reagents are all working ?
Hi Stephanie,
I agree with Paul's suggestion that you should include a positive control so that you know that the thermocycler is working correctly, as well as your PCR reagents. In addition, always include a negative control (no DNA template), so that you know that what you see is real and not some sort of DNA contamination. Was the positive band you saw previously quite intense? If it was really quite faint, then I wouldn't necessarily trust it. I would try and repeat some of the other colony samples that were loaded near what you think was the positive sample, just in case you have miscalculated which sample was correct.
I suggest to check samples DNA on agarose gel, follow the same steps as you did for the positive and got the band. Also include negative sample with no DNA. Hope you will get result.
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