Hallo, I am currently doing a project where i'm attempting to exchange a previously used polymerase in a PCR method, to Qiagens HotStarTaq Plus DNA Polymerase.
I worked very well on my controlsamples, and when I was happy with my results, I wanted to test out the new protocol on rutine samples. The results weren't as good as they were with my control samples. The homozygote samples have good clear and visibles lines on the gel, but the heterozygote samples, have only one visible line while the other is almost invisible. However only some of the heterozygote samples have this problem, other of them have two very clear and visible lines and look very good on the gel. So to me, it seems as if the samples have very different DNA concentrations, and so I'm wondering if anyone has any odeas to how I can fix this?
The PCR method has the additives DMSO and betaine if this helps. Also I tried to add both more and less sample to the PCR reations, and less was better, but it is still not enough to implement the new protocol with the new polymerase.
I hope you guys have some ideas
Dear Iben,
The trick I see, is that you would like to change PCR on samples with different DNA quantity, while using same method of amplicon detection - electrophoresis?
What I did, is actually to normalize DNA quantity or concentration over all samples, in the way the amplicons are seen well at electrophoresis.
You wrote that less DNA produced better PCR results - how you concluded that, if later you wrote it was not enough for the new protocol???
The one method to detect amplicons was electrophoresis, so if you concluded PCR was better is that you have seen the bands in the gel. So, what you would like still to improve???
The idea should be to normalize DNA quantity per well in order to have comparable results.
It surely work, but it might not be the solution in some cases. What is the purpose of the tests? Are you developing a diagnostic test based on agarose gel or blot detection?
Kind regards!!!
Apart from normalizing the concentration, confirm the specificity of heterozygous primers.
Alexei thank you for your response. The samples are tissue from mine tails, which have been lysed to get the DNA extracted. Because the tissue come in different sizes, there are different amounts of DNA in each sample. We test to see if the mice have a gene or not. If they are wildtype, WT, or knockout mice KO, or if they are both (Heteroxygot). For the ones who are just WT or KO mice, the bands show up clearly on the gel efter electrophoresis, but for the ones who are both, only one of the bands, the KO band, is clear and visible, while the WT band is almost invisible.
It is not possible for us to see how high the DNA concentration is in each of the samples.
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