I genotype and sequnce zebrafish using a bit of the fin. Lately though, I have been struggling with PCR. So far I have tried the following but to no avail:
1. Tried the primer pair in question with some control genomic DNA that I know to work. This has worked.
2. Checked the pH of my reagents. These are in place.
3. Re-extracted the genome from the animals that I'm having difficulties with and tried PCR with same primer set again. This has failed.
4. About to try the extracted genome with other primer pairs.
If the last step fails, what can I do differently? I extract the genomic DNA using proteinase K and NP40 lysis buffer, in a thermal cycler at 55C.
This is something I thought about already but it's just a subset of the animals that are resisting the PCR. I already know the external phenotype of the mutants as those don't even need to be sequenced because they are so distinctive. If my DNA did not have a matching sequence, I'd have thought it to be a mutation, which in turn would reflect on the phenotype. These animals have a single base pair nonsense mutation. Also, this is not the first batch of animals being genotyoped.
Hi Kunal,
Maybe you are using too much template DNA. If there is too much template, you generaly see a smear on the gel. Have you tried diluting the genomic DNA template 1:10 for the animals in question and then repeated the PCR?
Best Simone
I would like to express my opinion, though I have not yet studied about DNA polymerase. Since I like this kind of honest question very much, I particularly mention an opinion. I have personally informed about the defectiveness of PCR from honest Dr. Makoto Iwaya (an oldest member of the alumnae association "Wayu-Kai (in honor of the teacher Late Dr. Kazutomo Imahori, who firstly discovered thermo-stable enzymes), The University of Tokyo, Tokyo, Japan", and studied at Harvard University, Boston, MA, USA) that PCR is not applicable to infinitesimal amount of blood from the neonate. The velocity (V) of thermostable DNA polymerase reduces at high temperature, and accordingly Km of the enzyme may increase. I have found that the urea-treated lipoamidase of Lactobacillus casei (Shirota) shows larger Km and V values than those of the native (non-treated) enzyme (please see file; J Chrom B rat BIN LIP Km).
Further, Taq DNA polymerase purified from Thermus aquaticus (eubacterium) seems to be different from the DNA polymerase II large subunit of Thermoplasma acidophilum (archaeum). Therefore, species differences seems to also be very important issue in biochemistry (please see file again; J Chrom B rat BIN LIP Km).
Thank you Simone Schindler. Yes, I did try that too but to no avail. Also, this genomic DNA failed to give amplicons with other primers as well. However, I seem to have resolved the issue now. I think, and I'm not sure if this can be an interfering factor, the proteinase K I used for the extraction was off. I re-clipped the fins and extrated genomic DNA using a new batch of proteinase K. It worked for all of them.
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