It will be simple to clone your gene into pcDNA3.1 directly. Choose two cloning sites from pcDNA3.1, design primers that have each site on 5' end of one primer. digested PCR product with the two restriction enzyme, and ligate to pcDNA3.1. Just make sure there is no internal restriction site in your PCR product.
another way is to use blunt end ligation. choose enzyme like EcoRV or SmaI to cut vector, de-phosphorylate the vector, then ligate to your PCR product. In this case, you don't need to include cloning site in your primers. Just make sure your PCR is blunt end, not A-sticky end. Also, you have to confirm the cloning direction after screening.
Jun
The restriction sites and particular cloning strategy depend on the internal restriction sites (if any) present in the insert, whether you want to use the T7 and His tags of pET21, whether additional aminoacids are acceptable, etc. Can you provide more detail?
Bear in mind that pcDNA3.1 requires the gene to be brought along with its own Kozak and ATG, so if you insist on using the same enzyme pair for both pET21 and pcDNA3.1, you have to incorporate the Kozak+ATG after the site of the sense primer and make sure they remain in frame with the actual ATG of pET21 (the one in the Nde I site).
Hope it helps!
Hi there...
Cloning requires set of rules. Such guidelines are already mentioned on each enzyme suppliers (Such as NEB, Fermentas) website. For example use NEB cutter tool from the NEB website to decide which restrictions enzymes can be used for cloning. Use zero cutter option to select the enzyme of your interest.
Hope it helps.
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