I have run the PCR for several times getting the same light band results. I have also tried using more of DNA template but still the concentration is too little. Is there any mean to get more PCR product? How do I concentrate the DNA? I used recombineering protocol to insert my interested gene into chromosomes of E.coli.
Dear Anteneh,
How long is this DNA fragment?
When sequencing I try to send 50-100ng/ul of DNA but this depends on the company used. Usually when amplifying a PCR product 25-100 ng are totally fine.
Check the pimer pair and the temperature used for the annealing, also you can adjust the yield of the PCR with amounts of DMSO if primer melting temp are too high.
Hi Abebe,
To concentrate your product you can run 2 or 3 reactions in gel, elute it and use it as a template for PCR. In this way you can get band with more intensity which you can send for sequencing.
minimum is 10 ng required for Automated Sanger Sequencing and 100ng for plasmids
Dears All,
Many thanks for your concern. I used 20 cycles for 2.7kbp length PCR product and the annealing temperature was 64oc.
Dear Anteneh, if the polymerase system you are using is accurate and has proofreading activity you should go for 35-40 cycles to increase yield. I would test this before changing any other parameters.
Good luck!
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