maxi-cell procedures

Donald Chen - Microbiology chend at
Tue Apr 20 01:00:09 EST 1993


I'm trying to use the Silhavey procedure for maxicell preps to 
visulize proteins that are being made from plasmids that contain
inserts I am trying to characterize.  I have some questions about
parts of the procedure which I hope someone can answer.

1.  I understand that the UV dose is to nick the chromosomal DNA such that
most of the protein being made is from the plasmid which will then be labeled.
What sort of kill rate should I try for in order to achieve this result? Right
now I am using 190,000 microjoules (~300 ergs/mm2) and getting ~0.1% survival
(10 * 5 vs 10 * 8::survivors vs non-UV cells).

2.  This question is related to #1.  I just developed a test gel yesterday.
I have a number of bands which I have been told were from chromosomal proteins
being labeled.  Without going into a lot of detail, I have 4 lanes:a=wildtype
host with pBR, b=mutant host with pBR, c and d=recombinant pBR (with inserts).
Should I also include another lane for mutant host without pBR?  Is this
normally added in papers you have seen? Would this settle the question of 
many bands in my control and recombinant lanes?

3.  Does anyone know of a paper which shows the proteins I should expect
from pBR alone?  Or does anyone know how many proteins are to be expected?

Thanks for any help,


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