Sequences of PCANTAB Fragments cloned into pJET 11-11-11

2015-01-13

azim58 - Sequences of PCANTAB Fragments cloned into pJET 11-11-11


I now know for certain that the plasmid I have been working with is not
the PCANTAB5 E plasmid containing the insert described by Barbara Lipes
from Duke University. There are two major pieces of evidence for this.
Therefore, the plasmid we are working with is not the one we expected.
Either the lab from Duke sent us the wrong plasmid or our plasmid somehow
became switched in our lab.

  1. The plasmid is not the right size. The plasmid should be 4472+722 =
5194 (PCANTAB5 E backbone + insert). However, when the plasmid is
linearized and run on a gel the size is more around 8 kb.
see size of pcantab plasmid

  1. Sequencing results do not match as expected. The plasmid I've been
working with appears to contain thioredoxin and the LacI gene which are
not supposed to be present in my plasmid.
After this plasmid was still not behaving as expected, Andrey and I
finally just cut the plasmid into many fragments, cloned the fragments
into pJET, and sequenced these fragments. When comparing the contigs
assembled from these sequencing results, there was not one match with the
original PCANTAB5 E sequence or the insert that should be there as
indicated by Barbara Lipes.

The sequencing information was stored on 11-11-11

Contig 1 (204 bp)- overlaps with some sequence upstream of the LacI gene
in one plasmid on NCBI (Expression vector pNIC-H102)
Contig 2 (278 bp)- overlaps with part of thioredoxin gene in Escherichia
coli UMNK88

Contig 3 (687 bp)- overlaps with a non-annotated portion (685 bp) of
Expression vector pNIC-Bio3 plasmid pNIC-Bio3
Contig 4 (1134 bp)- overlaps with LacI gene (655 bp region (3579-4233) in
Co-expression vector pCDF-BIRA)
position of gene in vector: 3360-4442


Since I don't have the correct plasmid, I need to find a new plasmid to
use for my phage library.