Constructing PCANTAB rCompTT Plasmid

2015-01-13

azim58 - Constructing PCANTAB rCompTT Plasmid

I will put a "stuffer" into the PCANTAB plasmid so that I can well
separate a single cut plasmid from a double cut. Right now, I can't
separate a single from a double cut plasmid very well because there is
only a 40 bp difference between these two 4.7 kb plasmids.

To insert this stuffer, I can't use Sfii and NotI because SfiI has a lot
of "N" nucleotides that must match the ligated vector.

SfiI Site
NotI Site

Here is a picture of the sites near the SfiI and NotI site.

So I can use SacI or PstI. Andrey has some brand new fresh SacI

SacI Site
PstI Site

So now I just need to find a suitably large fragment from another plasmid
which doesn't have any origins of replication or any other potentially
bad things in it. This fragment needs to be cut out with NotI, SacI, or
PstI.

pet32 has a SacI and PstI but it also includes an F1 ori
pGEX has a PstI, but no others.
pCMVi-LS has SacI and PstI (1347-722) = 625
pCMVils-rCompTT only has NotI

I'll use the pCMVi-LS then

Cutting with PstI and SacI yielded two bands with the PCANTAB

surprisingly. I think I may not have the correct sequence for the
PCANTAB plasmid.

I think I may need to find a stuffer with SacI and NotI

o However, before I start searching for a stuffer, I should make
sure that cutting PCANTAB with SacI and NotI does not drop out any
large fragments as PstI and SacI did.

I found a plasmid with a stuffer that will work with SacI and NotI. See
Constructing PCANTAB-pRSET plasmid