I am curious to know that in case we plan to add a ~25 base pairs long site in our vector , how can we do that. We do not have any template for the given site so I was wondering whether we can get the double stranded DNA synthesised together with particular restriction sites and then using PCR we can increase it's concentration. After doing that we can normally follow the restriction digestion and ligation protocol.
But the main problem is that can we use 25 bp long DNA fragment as template in PCR? Or, alternatively how can we add a new site in our vector if we do not have the template with us that might help in PCR of the site of interest ?
I would like to suggest you Polymerase Incomplete Primer Extension method where you have to just design primers with the bases you want to add and just do one round of PCR and do transformation to get desired insert in your plasmid. Please find attached paper which will help you to design your experiment. I have added TEV protease site at N-terminal of my protein which was 21 base pair successfully. Hope this will help you.
Thanks Mrityunjay for your kind suggestion, but I am not able to find any attachment. Please can you attach the paper once again because it would be of great help to me.
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