Hello! I would like to know how can I amplify two fragments: 340bp and 1500 bp with the same primers set.
I have designed F and R primers to amplify two fragments, but the result is just the amplification of the 340 bp fragment. How can I improve the amplification of the 1500 bp fragment,
obs: I've tried using DMSO 5%, but I have very little product of this fragment.
Thanks a lot for your help!
Primers will anneal almost anywhere if the annealing temperature is low enough. The best primers for amplifying long amplimers are long primers so that annealing takes place at high temperature so high specificity.
You can go up to a maximum of 10%dmsa and also add a final concentartion of 1Molar betaine because longer amplimers may have secondary structure problems and dmso and betaine minimise these structures and the betaine will improve the dna melting step of the pcr cycle. Do not use too much primer or it will anneal in other places. Use a short annealing time of 10 seconds and increase the extension time to 2 minutes to make sure that there is plenty of time for the polymerase to fall off and another polymerase molecule to anneal and complete the extension. Run an increasing temperature gradient to try to maximise the longer pcr product.
Primer blast your primers and if there are 2 sequences with perfect priming then redesogning new primers or re amplifying one band at a dilution of about 1 in 1000000 and 20-25 cycles may give you plenty of product.
The amplification of the smaller product will always be more efficient than the amplification of the larger product.
As Paul Rutland said, you can increase the extension time to give the larger product more time to build.
Or you can design new primers, ones that are specific to the 1500bp product & ones that are specific to the 340 bp product and set up separate PCR reactions.
Good luck!
Thanks a lot for your suggestions Paul Rutland , I gonna try with Betaine.
The conditions of the PCR where I have at least a bit of amplification of the major product are:
60ºC Annealing
5% DMSO
I hope Betaine can improve the product of 1500bp.
Thanks
Good luck Cleidy Mirela Osorio Mogollón I would use both 5%dmso and betaine especially if your pcr product is GC rich
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