Is this randomly decided by the researcher or is there a general principle they can base their decision on? I have read many papers looking for an answer but have yet to find an explanation on preliminary tests. I'm trying to determine which pairs are the best in terms of inter-population polymorphism and amplification success and clarity.
I think it depends on the system and the level of screening. If you are just starting out and want to know how many base-pairs to have on your overhangs, you don't need a lot of samples. When you're getting down to the exact primer pairs, it depends on your question. If you are looking at a lot of populations, I would say have at least 1-2 per population. If you are looking in one population over something like a hybrid zone, you should have samples from each parental area and ones from the putative zone. I always like to structure tests based on plates as well, making sure to have at least one repeated sample and a negative control for each step (construct, preamp, selamp) to check repeatability, etc. This usually results in 3-4 primer pair combos per plate for optimization purposes.
You seem to be concerned more with population level variation, so it would be best to screen a few individuals (as the above answer indicates) from each population to ensure a representative spread in the potential variation to be detected. Definitely run the repeats/positive controls and a few blanks with each run too.
Sam,
I you are using a bi-parental mapping population then you should screen the parents, since it will be these data that you use to construct your allele panel for the pop screen.
" I'm trying to determine which pairs are the best in terms of inter-population polymorphism and amplification success and clarity."
If you have the same start primer eg AA then you could try say 10 combinations but it will depend on your time constraints, and your funds. e.g. EcoR1 (E+AA) and Msel (M+CAA, CAC, CAG, CAT, CTA,CTC, CTG, CCT, CTT) try the combinations then count the number of polymorphic loci. The selection can then be based on the amount of information gained. Using a range will give greater coverage across the genome. If you are using different pops then follow pretty much what Julie and Jasmine recommend.
Be careful with the starting DNA concentration, i.e. have your stock to at least 100ng ul.
I would suggest you to have some literatures reviewed on primers combination as it depends on crop to crop. General hint specific to crop can be obtained from AFLP primers kits. However, determining the number of primer pairs would depend on your objectives as well. Based on literature specific to species, you can select the combinations that has given high rate of polymorphism and then depending on time availability and your objectives you may use about 5 to 10 primers combinations
One has to run an EXPLORER with a prototype / representative sample to ascertain the primer pairs that are responding optimally with the genome of the species under consideration. There is no rule of thumb as such.
Hi, like every one has said, it depends on what your focus is, for me i was looking a just one particular species across different population aín different countries to i took 2 samples from each population in each country this will give you a better results, even though the species are the same they are far apart. I have screen a total of 64 combinations of the Selective primers and out of which i did choose 6 that gave best results for futher analysis with my entire samples.
Dear Collegue,
to a a much beter result than any other up to now you should take a sample of 30 pairs andtha make your calculationswith n-1.
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