Hello everyone, I hope you will understand what I want to ask. For exemple: I have 500 genomic DNA samples (different genotypes of same plant species) and I have 10 pairs of primers (Fwr+Rwr). I would like to genotype all my samples (500 samples), with the most efficient primers (maybe 4 or 5). Because those primers are going to be labeled with fluorescent dye (that is expensive) and due to lack of time, I would like to use only those primers, who are specifically and efficiency for sure.
So is there any percent of all samples that should be checked on agarose gel or sequenced before genotyping all the samples only with the best primers (4 or 5) on polyacrylamide gel ?
1 DNA sample. You test all your 10 primers in 1 DNA sample and see which one works best (in agarose gel).
Reason: if it doesn't work in other DNA samples, it will mean that your gene (piece of gene) is not there. You need only 1 positive control to be able to say "these primers work".
Thank you Yulia. Basically we already checked 10 primers on 20 DNA samples. It looks like all primers working but not on every genotype. Because those primers was constructed to work only on chloroplast DNA, we sequenced 3 genotypes with those 10 primers to be shore that they "working" on cpDNA but not on nDNA. I hope this is enough to start genotyping...
In this context, you should maybe use a standard of pooled template (RNA/cDNA/DNA, possibly from one donor or donors of known different genotypes ich you want to detect snips or other differences) which can be used to test the different primer pairs. Using this evaluation basis you can decide which pair is the most adequate.
Thank you Andreas. We just want to find out is there any polymorphism between different genotypes based on chloroplast genome analysis. It should be mentioned that our research subject is crossbred plant.
To detect snips would be perfect, unfortunately it cost a lot with such amount of samples and primers... That's why we would like to genotype samples on polyacrylamide gel
do you already have these labelled primers?
I ask because if they are being analysed on a capillary sequencer then with 4 different coloured dyes and maybe 4 different sized amplimers you can load 16 different amplimers in each capillary position and then the cost of a 96well analysis will be quite cheap per sample as each well gives 10( in your case) results and companies usually charge for a plate of samples and the number of samples in each plate and position is not usually important....it is just the set up charge that determines how much you will pay
Dear Mr. Paul, thank you for your response.
We have 5 labeled primers of 10 (labeled IRD 800 because we are going to use Li-cor 4300 analyser for microsatellite analysis). I really like an idea to do multiplex PCR or use amplicons for NGS, we will consider that...
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- DNA