I used KAPA hifi pcr kit to linearize the plasmid for in-fusion cloning.
Following the protocol, the DNA template amount was 5 ng and I already diluted it 100x. Annealing temperature: 66 69.
The primer was designed on TAKARA website.
Gel electrophoresis method: 120V 60min
5ng of a very small template is a huge number of molecules, i think that there is too much template and possibly too many pcr cycles, Every 10 cycles is 1000 times more pcr product, Aim for about 30,000 molecules as a starting template and set up 4 tubes of pcr and remove one tube at 20, 25 30 and 35 cycles to get an estimate of where you get strong,clean amplification, Your gel looks like great over amplification where there is so much product that you are getting product annealing with other product molecules and creating longer concatamers
I would first check the quality of my plasmid prep; do you have some cell DNA in it ? also your annealing T° seems quite high (55-60°C is standard). maybe look at your elongation time (30sec/kb) maybe the smear come from unfinished PCR products
Didier Poncet Hi Didier, sorry for the late reply.
The plasmid was extracted from bacteria culture by miniprep. OD value and Conc. of the plasmid is quite normal. I am also thinking the annealing T was too high but it worked previously for the other plasmids. What do you mean by unfinished PCR products?
Thank you for your answer!
I mean that the polymerase has not finished all the product which result in partially or totaly single stranded DNA. I absolutally do not trust the OD of miniprep; there is frequently small DNA and RNA fragments even oligos that you can't see on a gel. put undigested (and undigested incubated in buffer without restriction enzyme and incubated in buffer with restriction enzyme) on a gel to check the quality of your prep. or start with a maxi prep...
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