Hi,
I'm pretty new to PCR/genotyping - I ran an experiment recently using a protocol that had been done by a previous lab member. I was expecting to see the knock out band and WT bands amplified in all of my samples, which I did, but the knock out band was very faint in the experimental samples. My controls looked normal. I also had primer dimers in my experimental samples, which didn't show up in the positive or negative control. I'm hoping to optimize and re run the experiment soon, but I'm not sure what the best course of action is. Should I increase/decrease primer concentration? Is there something I could add to optimize my results?
What was the original concentration of DNA going into the reaction?
I'd quantify the DNA for ng/uL and target 25- 50 ng/uL. If that doesn't work look at the primer concentration.
Hi Haley,
in addition to what Michael Shawn Hooker mentioned you might also consider the quality of your template (as it seems that your positive control looks good).
I don't know which protocol you use, however if the DNA is not clean enough the PCR might not work optimal (and this might be different for the wildtype and the knock out so that the wild type PCR is still working fine but not the knock out PCR).
I have done a lot of genotyping myself and I know that several protocols involve proteinase K treatment with heat inactivation, but with this approach you can have some residual proteinase K activity what can also cause problems.
So if you still have problems after following Michaels recommendations and you are not doing so already I would consider Phenol-Chloroform extraction to clean up your DNA (after proteinase K treatment).
KR Dörthe
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