I have insert having flanking regions with XbaI site and and a vector digested with XbaI. How can I prevent self ligation in insert as well as vector before ligation?
Hello! So this not ideal. However, this is how people used to do it all the time. You will most likely need to treat either the vector or the insert (ideally the vector) with alkaline phosphatase. This will prevent self ligation. I would suggest using highly competent DH5Alpha E. coli like Invitogen top10. IPTG/xgal plates will help if the vector supports this. Hopefully it's not a blunt end restriction enzyme because you can have orientation issues. Here is the NEB article on AP. Good luck!! https://www.neb.com/en-us/applications/cloning-and-synthetic-biology/dna-end-modification/dephosphorylation
Dear Moein Iranmanesh
as Matthew Edward Thornton told it is not ideal since, even if you use CIP treatment of the vector to reduce the probability of self ligation, you will have random and no directional insertion of the fragment and therefore after you need to design and set up a good PCR colony screening that is able to identify the colony where the insert carry the right orientation.
Hovever is suggest to you to skip the use of restriction enzimes and use the PIPE cloning approach. You need just an high fidelity polimerase, to amplify both vector and insert, design primers with overhang extremities and a specific e.coli strain (mach 1 thermo work well)
If you are interested to know more details about it you can read the following papers of the inventors of PIPE:
https://pubmed.ncbi.nlm.nih.gov/18988020/
https://pubmed.ncbi.nlm.nih.gov/18004753/
and/or give a look to the following videos on my blog (ProteoCool)
https://www.blogger.com/video.g?token=AD6v5dymTB4mmANTKeTOyzXQq7QSUurTLQFPDsGxi4FqyNDpu3YZutcuN0VfLTfDTPRD8edEIDPD6bJZIe_VWirwCNZHkJ1A34BATcDIzXZEbV1UUtRLR6Nc19tU2_t6-Or5Sag2dwU
https://www.blogger.com/video.g?token=AD6v5dx7hIjOrRfLt70Lph2VJXXgyfu7CVeVUqMIzPNb5ySq3fHJF5QSXLTq93C-8iRHI_fT9_JfcwYQiGCHj0uYBMQzkB7DU8_qi34YSr9ZJzA7OYfHQav5-9eO6Idz5f06bydovl8
good luck
Manuele