subcloning fragments - cloning (Aug/28/2005 )
hello,
i need to subclone a gene into a new plasmid. the problem is that it does not have unique restrictions sites at each end. therefore i am thinking i will have to cut the gene into 3 fragments. has anyone ever done this before? is it a fairly easy procedure with high success rate? can you offer any suggestions that may help me out when i do this?
many thanks!
It should work, you might have to gel purify your vector before ligation.
Why don't you just do PCR of the gene?
Attach a restriction enzyme on each primer, if you want.
It's quite easy, but if this is the first time to do PCR for you, read the cautions before you do it. (Primer design etc.)
That would be much better.
Attach a restriction enzyme on each primer, if you want.
It's quite easy, but if this is the first time to do PCR for you, read the cautions before you do it. (Primer design etc.)
That would be much better.
i didn't consider simply PCRing the gene because it is very long (ie >8Kb)... maybe this is a possiblity with the new long-template pcr systems... what do you think? thanks.
What do you plan on doing with the subcloned gene? You may want to look into a high-fidelity Taq polymerase (ex. Phusion Polymerase from NEB) if you plan on expressing protein.
Also, if you plan on subcloning by introducing restriction sites with primers, don't forget to add the appropriate number of bases upstream of your restriction sites.
-Hank
Amplification of about 8kb DNA was okay for me with TAKARA LA-Taq. Maybe there are many commercial products for long PCR.
Of course it's more difficult than normal PCR and it's dependent of the DNA to be amplified but it's possible anyway.
Look up the cautions from Molecular Cloning or other textbook, before you do.
Generally, you should set the Tms of both primers as equal as possible,
and the length of the primers should be at least 25mers (Tm=60C),
and extention time should be longer than usual. (1 min/kb DNA).
Good luck!