the Peb1 saga continues.
i grew up my three plates of E. coli:
P2x, c2x, and pMAL p2x
and they looked good (got isolation!)
so they get wrapped up and put in the fridge until we can mini prep/pcr.
we are going to run them side by side with unmodified p2x and c2x to verify.
the cut sizes are:
p2x (6721bp) cuts with PvuII at 1032, 1125, 2898 and 6077 (not including insert)
c2x (6646 bp) cuts with PvuII at 1032, 1125, 2823 and 6002 (not including insert)
and herein lies a problem....
the base pairs are so close will we be able to see the differences on the gel? there's only a difference of 75bp in the third cut and 75 bp in the last cut.
each base pair is an alphabet (you know, dna is made up of the letters GTAC). 75 letters. not much at all.
suppose we can't see it even on a 3% gel. how can we tell them apart?
so we've been shopping for ideas- other ways we can compare- in case the gel doesn't work.
and that is when i realized i had misunderstood the pMAL. i thought it was FROM campy, but in fact it is the plasmid from E. coli.
it is a plasmid from E. coli and can be inserted into E. coli and is able to be modified.
we are modifying it (at the "multirestriction site"/cloning site) with Peb1. we are sticking peb1 into the pMAL plasmid allowing E. coli to clone it for us!
so, pardon my mistake in the previous post, where i had said that p2x comes from campy. it is the peb1 that comes from campy (in 2 forms periplasm: p2x or cytoplasm c2x).
at least i think so. man, it's hard to digest all of this.
here's a map of pMAL p2x of E. coli
and if anyoneeeeeeeeeeeee has ideas how we can compare the two plasmids, i'm eternally greatful!
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