cloning into Pgem t easy - (Apr/26/2012 )
hi all ,
im quite new to the whole cloning thing and i bet my question sounds stupid but here it is :
i need to clone a set of genes and from what i heard it seems that i need to clone it first into pgem t easy vector assemble the genes there and then subclone the genes into my expression vector. why do i need to clone it in pgem t easy first? why pgem t easy?
You don't have to clone into the pGEM-T easy vector first, you can easily clone straight into your expression vector, but it is a bit more of an involved process:
As you are presumably cloning by PCR, normal Taq based PCR results in a single bp A overhang on the end of your sequence, this can be used to clone into T-vectors such as pGEM-T where the vector comes pre-cut with a T overhang and DNA ligase conjugated to the ends, which makes cloning quick and easy. This is known as T/A cloning. The downside is that you have no control over which direction your gene will insert, so you need to screen for direction. It is also possible to get expression constructs such as pcDNA-3 (or higher) which use this system.
However, you can also just design restriction sites onto the 5' end of your primers, and amplify, then digest the product and vector with the appropriate restriction enzyme and ligate. You could also just blunt-end clone (get primers with a 5' phosphate), but this has the same problems as T/A cloning.