Hi Researchers,
I am seeking guidance on how to delete an existing restriction enzyme site and insert a new one in the pcDNA plasmid.
My understanding is that I would perform PCR using primers designed to delete the unwanted enzyme site without including the sequence of the enzyme I want to remove. After this, would I then transform the plasmid and carry out another PCR with primers designed to include the sequence of the enzyme I want to insert? Would this second transformation represent my final result?
I would appreciate any support or alternative techniques you can suggest.
Thanks,
Aabid
Dear Aabid Hussain Ph.D
it depend if you would like to replace the new site in the same position of the old site. theoretically you can just replace the old site with the neww with a single PCR where you amplify the entire vector with primers annealing just around the old silte and carryng a flanking region that contain the new site and overlapping.
To undertand better it, read about PIPE cloning in
Article Combining the polymerase incomplete primer extension method ...
or look to the following posts
https://www.blogger.com/video.g?token=AD6v5dx7hIjOrRfLt70Lph2VJXXgyfu7CVeVUqMIzPNb5ySq3fHJF5QSXLTq93C-8iRHI_fT9_JfcwYQiGCHj0uYBMQzkB7DU8_qi34YSr9ZJzA7OYfHQav5-9eO6Idz5f06bydovl8
https://proteocool.blogspot.com/2021/09/tips-of-week12-rapid.html
on my blog
best
Manuele
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