Constructing+PCANTAB-pRSET+plasmid

We are constructing the PCANTAB-pRSET plasmid so that we have a larger stuffer that can be cut out and help us separate single cut from double cut PCANTAB plasmid.

The plasmid containing our stuffer is E183L in pRSET Nat GFP (I'll just refer to it as pRSET). The stuffer will be cut out with SacI and NotI. It will also be inserted into PCANTAB in the reverse orientation so there are no worries that it will translate into something with a normal structure that could interfere with other things.
 * At the time the plasmid was in slot 25 of one of Mark's boxes (U54 FTU/ASFV genes (full-length) sequence confirmed) (260 ng/uL)

Sequence of stuffer from E183L in pRSET Nat GFP This is the SacI-NotI fragment (with entire restriction site shown). Lowercase is the reverse complement of part of rGFP, and uppercase is reverse complement of part of ASFV gene E183L.

Gel of pRSET and PCANTAB Cut with SacI and NotI see 9-24-11 of notebook for details

Note: a sequential digest was necessary for this digest to work effectively

Sample Order: 1 kb NEB, Double Cut PCANTAB, Double Cut pRSET, NotI PCANTAB, NotI pRSET, SacI PCANTAB, 100 bp GeneRuler Plus



Note: For some reason the single cut samples look quite smeared. I think this is because I may not have loaded the samples all the way down into the large deep wells I used.

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Transformation Results

PCANTAB pRSET Ligation



PCANTAB Ligation Background