expression problem in BL21(DE3)
ekhatipoREMOVE at midway.uchicagoREMOVE.edu
Tue Mar 26 14:50:40 EST 2002
BL21(DE3) are not resistent to Cm. You probably inedvertantly switched the
stocks. However, pLysS cells should be very easy to lyse after
freeze-thawing, unlike original BL21(DE3). If you cells don't seem to be
autolysing too easy, could that be that they have another pACYC plasmid, for
example pACYC-RIL or pACYC-RP to deal with codon bias issue.
"Kyle Legate" <legatek at mcmail.cis.mcmaster.ca> wrote in message
news:Pine.SOL.4.33.0203261000070.8014-100000 at mcmail.cis.mcmaster.ca...
> Hi all, I have a problem I hope someone can help me with. I'm trying to
> express two proteins in BL21(DE3), one on a high copy vector under Amp
> selection and one on a pACYC vector under Cm selection. I get great
> expression from the high copy vector but nothing from the low copy vector.
> I tried plating cells transformed with only the Amp plasmid on Amp/Cm
> plates (100ug/ml amp and 70ug/ml Cm) and noticed to my dismay that the
> cells grow just fine! I was under the impression that only cells
> harbouring pLys were supposed to be Cm resistant--am I wrong? I'm
> beginning to wonder if our DE3 stocks became switched with our pLys
> stocks, which are supposed to be stored in a different box--not good since
> I won't be able to transform them with pACYC.
> Also, do BL21(DE3) harbour any intrinsic antibiotic resistance?
> I'm starting to clone into a tet resistance vector but don't want to
> continue if BL21 are resistant to tet.
> Thanks for your help.
> ... . . . . . . . . . . . . . . .
> legatek at mcmaster.ca Kyle Legate legatek at hotmail.com
> Tower of Tongues:Thursday PM:10:30-11:30 EDT:http://cfmu.mcmaster.ca
> moon musick:ritual:IDM:experimental(electronica):minimalism:glitch
> . . . . . . . . . . . . . . . ...
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