help with His-tagged vector construct - (Feb/28/2008 )

Hi, for the past couple of months ive been trying to make a His-tagged protein with TEV protease recognition site using pET22b+ vector and two linkers (both about 60 bp) where I substitute the XbaI-HindIII portion of the MCS of pET22b+ (contains cut sites I dont need) with a simple XbaI-RBS-NcoI-HindIII linker. My gene contains an NdeI site, so I cant use that as the start. I also wanna add a linker to the HindIII-XhoI site of the thus modified vector that includes a TEV protease recognition site (i.e. HindIII-TEV-XhoI). I got bogged down in the linker digestion/ligation, where I cant even seem to get the linker (the latter one) ligated into the vector after RE digestion (according to analytical digestion with same enzymes), although I get transformants each time (I use TOP10 cells which will transform with even one intact plasmid in there). At the moment I get a double size vector band (overloaded so difficult to see) and I tried digesting a larger part off (XhoI-MluI), but it produced bands of about 2500 and a very large band (assumed to be double or triple vector). I tried dephosphorylating the vector and then ligating with 1/10 diluted vector with 100x more concentrated insert, but it remains to be seen what this will do. So I dont know what to do at this point. Anyone have any pointers with regard to linker ligation into vectors?

I’ve made a few MCS and inserted them into vectors. There are a couple ways you can try to do this. The way I did it was as follows:

1) Buy two primers that, when annealed together, have overhangs complimentary to the sticky ends you will generate in your vector when you do your digest.
2) Mix them in a 1:1 molar ratio, heat to 95C for about 5 minutes, and then cool on ice for another 5.
3) Digest your vector, run it on a gel and isolate the plasmid band.
4) Ligate the digested vector with the annealed primer pair (remember they have overhanging sticky ends).
5) Transform this to your cells.

This saves you from having to digest the linker and losing some of it during the purification step.


Your primer pair would look something like this:


Fwd Primer:

5’-AGCTT–TEV Sequence – C-3’

Reverse Primer:

5’-TCGAG–TEV compliment–A-3’


When you anneal them you would get the following product with pre-made overhanging sticky ends
5'-AGCTT–TEV-C-Xho-3’
3’Hind-A–TEV-GAGCT 5’

Thanks.

Yeah I tried this but it doesnt work.