I used to genotype my KO animals following this protocol: I extracted the DNA from the tail using a Sigma reagent and I performed the PCR reaction with a Promega Green MIX. It worked well, but due to the high costs we are trying to migrate to a cheaper option which I'm having trouble to make work for one strand specifically.
I changed the extraction reagent for the Hot Shot reagents (lab made) and for the PCR MIX I'm using the Gene Direx reagents (a cheaper chinese brand). It worked totally fine for one of my other mouse strain, but for this strain in particular (picture attached) I'm having those smear bands only with the samples I extracted with the Hot Shot reagent. The first 2 lanes I used a sample extracted with Sigma reagent and the bands are (kind of) clear, but for the other lanes (DNA extracted with hot shot) there is only smear or nothing at all. I tried adding more DNA and/or more MgCl2 but it didn't work. Can someone give me a tip on this one? Thanks in advance.
Just an update, I used the same DNA templates of the previous gel and performed the reaction with progema green enzyme. As you can see on the picture below, it worked for my DNA templates extracted with the hot shot method, therefore I believe the DNA template was not the issue on my previous gel (except for 1 sample that didn't appear on both tests). It seems to me that it is happening due to some incompatibility between the hot shot reagent and the Gene Direx enzyme, but this is specific for this mouse strand, since it works for other genotypes. I wonder if there's a way to make them both work together for this specific gene.
PS: this 2nd gel I ran in another apparatus and it ran more slowly. I don't know if that would affect anything.
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts