Changing the vector - (Aug/05/2014 )
I need an advice where to start and what point need to be taken in advice. I have a gene that needs to be moved from vector A and inserted in vector B.
there is no similarity in the cloning site in terms of restriction enzymes.
Thank you in advance,
Amplify the gene with primers that contain the restriction sites you want to use.
It is 4kb gene. Do you think I can do that by Primer amplification ?
Yes. This should be routine, with sufficient extension time.
Make sure the restriction sites are at the 5' end of the primers. Also ensure that you have 6 bp (any sequence will do) 5' of this, so as to give the RE something to bind to.
Primers should look like this:
Ok! Thank you very much bob. I will be inserting This gene into pTRE3G vector . According to the MCS I can use 11 restriction enzymes. Is there is any preference? What is the most easier tool/ program to use to design the primers? (I am mac user)
Use enzymes you've heard of before and that you already have in your freezer. Choose ones that can be heat killed to save you a purification step after cutting. EcoRI, PstI, XhoI, HindIII, XbaI, KpnI
Thank you indeed, but non of these enzymes in MCS I will google for my options. What about primer design tool ? How long the minimum sequence overlap between the primers and the gene that need to amplify ?
I would go for BamHI and BglII, neither can be heat inactivated so you will have to clean up after the reaction, but they should work in the same buffer (at least NEB tells me so) so a double digest is a possibility.