I have a PCR which generates multiple PCR products of different sizes from bacteria with one primer pair in one reaction (PCR-ribotyping). The PCR products are between 150 and 600bp but I'm getting much more amplification of the small products than the bigger products. I'd like to reduce the amplification of the small products and increase the amplification of the bigger products to get a more even fingerprint pattern.
Am going to try increasing extension time from 1 min to 2 min and have various other standard PCR optimising things to try but if anyone has any specific ideas I'd love to hear them!
Well, that isn't real AFLP reaction, but as on AFLP in order to decrease the number of bands, add a extra base to your primer…
well, choose the 4 possibility … see which one is better…
but if you keep the same primer the an increase of elongation time is the best way, indeed if your time is too shor the big produce won't be finished the not reamplified in the next cycle etc…
Thanks Benjamin.
I know it's not real AFLP but the result is similar.
Can't change my primers unfortunately.
Well then just increase elongation time …
or…
Well one on the reason why you may have fewer amplification could be that the temperature needed to denature biggest amplification produces is slightly higher and maybe they do not melt correctly and then ain't amplified correctly…
If you have chemical and time to waste, it deserve a try…
Anyway if you try it, I'd like to know if you see any differance ;)
You can also try to reduce the amount of template DNA. To much template during the first cycles might cause competition between templates with primers, and the shorter fragments might be overamplified.
If you are using a protocol where you start cutting your DNA with 2 restriction enzymes and you use a frequent cutting enzyme and a rare cutting enzyme and your primers are adapted to the restriction sites, you get 3 kinds of amplified fragments. a lot of small fragments with both sides of the frequent cutting enzym and 1 of the primers annealing on both ends of the DNA fragment. Larger fragments with one side the rare cutter and the other side the frequent cutter, using both primers for amplification. And very few large fragments with both end cut by the infrequent cutter and the other primer annealind to each end of the DNA fragment. So I don't think increasing extension time will help at all, the smaller fragments are in bulk present. I think gel running conditions can help to get small fragments run together as a few bands while the bigger fragments still run as seperate bands or as in AFLP label your primer for the infrequent restriction site and if you only visualise labeled fragments you won't see all the very small fragments. I hope this give you new ideas to continue.
T v.d. Reijden
Hi Tanny. There is no restriction involved. It's not traditional AFLP. It's just something that gives a similar kind of fingerprint.
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