I did AFLP with fluorescent primers by Applied Biosystems ( EcoRI-ACT-FAM(402045); EcoRI-AGG-JOE (402043); EcoRI-AGC-NED (4303057)). I would like to continue my work, but primers have been discontinued!
There are two ways: 1) synthesis of primer ... but if I let synthesize primers, they can vary in concentration and purity. The problem is in the original fluorescent colors too (it is not easy to obtain them). I´m afraid, that It will be impossible to join the old and new result (?!). Do you know which firm did synthesis of primers for Applied Biosystems?
2) somebody (firm, lab, person) have old stock of primers... but how I can find him? Is some "market" of unwanted stocks?
Have anyone the same problem? How did you solve it?
I'd just go with synthesis. ThermoFisher (previously Applied Biosystems and Invitrogen) still sells FAM and NED, but not JOE. Eurofins MWG Operon (www.operon.com) sells all three. Both are good labs with high purity. The only way to be confident about joining your old and new results is to try the new oligos with the old samples (duplicate your previous work in a few diverse genotypes) and compare with your old results. If your markers aren't reproducible, they weren't very good markers anyway.
Old primer stocks can degrade. If you are worried about reproducing your results, I would think that using old primers would be more risky than using newly synthesized ones.
Best of luck with your research!
I agree with Jack, you should go for the synthesis of new stocks and re-run several of the old samples with the new stocks. Then compare the new peak calls with the previous ones ... this will help you to see if shifts happen in the peak patterns. You can then consider those shifts in your further analysis. I had to go trough a similar procedure with a microsatellite study which was done in two different labs. It's quite a mission to make sure that everything is comparable, but with a bit effort it's doable. However, AFLPs might be a bit more difficult.
Good luck.
Thank you very much. It seems good way. However, Operon don't sell NED colour. I will have to combinate Fisher and Operon.
I´m not shure, but I think, that Applied Biosynthesis used 5-FAM as fluorescent colours in my primer. Do you think that is differences between 5-FAM and 6-FAM?
Ah, sorry I didn't notice Operon lacks NED.
I'm not 100% certain how the differential placement of the -COOH on 6-FAM vs 5-FAM would affect migration of your product, but I suspect that it would be minor and would probably only shift your bands in one direction or the other, if anything. That would probably be a good question for a representative of one of the these companies. If you know for certain which you used previously, Operon offers either 5-FAM or 6-FAM.
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