I want to amplify a gene whose end terminus sequence is in such a way that the after designing NotI restriction site inserting (reverse) primer; it forms primer dimer during PCR amplification. I have tried gradient PCR, touchdown PCR; still facing same problem.
Alternatively, I have gone through the protein sequence and tried to change codon sequences corresponding to each amino acid resulting different sequence; solved the primer dimer formation due to end terminus sequence but got new primer dimer formation due to NotI I site. Since, this amplification is for secondary cloning purpose..I can't change the restriction site.
End terminus protein sequence- DVAPH and DVAPHC
Primer needs addition of NotI site and 8 bp random sequence.
Thank you.
look man, first of all make sure that your primer concentration is not much higher.. because at high concentration of primer it may forms dimer instead of binding to the specific region. and most importantly try to do PCR in 3-5 degree bellow the annealing temperature of the primer. In that case you can run a gradient.
Mr Kaustav, I have tried it by reducing (half than the standard) primer concentration, adding DMSO up to 5%, using Phusion polymerase and purified template DNA.... still persist the same problem.
use a hot start polymerase then the temperature may not drop low enough to make a primer dimer. Alternatively set up the pcr without enzyme ,hold it at 80c then add the enzyme and start cycling immediately for a hot start. You can still do either a gradient or touch down pcr using this method of hot start
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