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
 * 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