I had started a thread a little while back about my adeno-plasmid from hell that was
22kb and refused to grow stabily in Dh5a or DH10b E. coli cells. It would rearrange
when grown in large volume (250 mls or more)
Well upon the advice of many people who responded to my post I went and shelled
out 230 dollars canadian (`150 american) for "sure" cells from Stratagene. I got
five tubes at 80 ul of bacteria per tube enough for 10 electroporations.
I electroporated DNA from the first time I isolated my plasmid (by hybridization by the way : ) )
I got an amazing number of transformants, approaching 10 to the 10!
So far so good. I preped 10 colonies all were the right size and when digested with
various enzymes gave my the right bands. The Dh10 and DH5 cell had given only
around 5 out of 6 or 7. I then decided to try a few things....
1. I innoculated 5 mls of LB (with amp 50 ug/ml) with a single colony from a previous
broth when I initial prepped the transformants
Result: no bacterial growth and no DNA!!!
2. " " " with a single colony from a streaking of an original
transformant.
Result: a moderate amount of DNA (1ml gave me about 1 ug of DNA)
3. " " with 100 ul of an initial LB broth used to prep the transformants
Result: lots of bacteria/ large RNA/DNA pellet.... less than 15 ng total DNA : (
4. " " " with 100 ul of frozen stock of one of the positive transformants
Result: lots of bacteria and large pellet .... less than 10 ng of total DNA
5. Lastly for the hell of it I took one colony from a restreak of one of the original transformants
and dropped the toothpick and all into a 1 l flask with 250 mls of LB /amp....
Result: less bacteria (od) than 3 or 4 and smaller pellet but in 1ml 2 ug of DNA
I prepped the rest of the DNA and I got 280 ug of DNA from the 250 mls of culture. It is the right
moleculare weight ect.
one interesting thing is that the DNA exists as oc and cc form and when you cut with enzymes
some DNA remains uncut in a highly negatively supercoiled form (aprox. 1-5%). I think this
may be due to the fact "Sure" cells lack gyrase but any way it may also partly be responsible
for the lower recombination. (either by limiting toxic gene products from being trascribed from
the plasmid or by preventing access to recombinatory proteins (what ones are left <grin> )
thanks again to everyone who answered my post
G.
_______________________________________________________________________
Graham Dellaire Snail Mail:
Red Cross, Research
McGill Univeristy Montreal Blood Services
Faculty of Medicine 3131 Sherbrooke St. East
Div. of Experimental Medicine Montreal, QC, Canada
E-mail: popa0206 at po-box.mcgill.ca H1W 1B2
B2XE at musicb.mcgill.ca
Fax: (514) 525 0881
Voice: (514) 527 1501 ext 175
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