expression construct question

StewJW stewjw at gmail.com
Fri May 26 09:42:06 EST 2006


"You can do either. Invitrogen do a bicistronic vector (pBud something
or
other). With it you get a first MCS under CMV promoter and a second one

under a hEF1 promoter. Works OK but is quite expensive,and uses zeocin
as a selectable marker which is crap and very expensive. "

pBudCE4.1 Available from Fisher VXV53220

Uses CMV promotor with optional -myc epitope tag for rapid detaection
and 6xHis, and EF1-alpha promotor with optional V5 epitope tag and and
6xHis for purification. The link below contains useful references:

https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=16006&CMP=LEC-GCMSSEARCH&HQS=pBudCE4

Zeocin is used as the selection gene and is used successfully for many
Invitrogen products. E. coli strains that encode the ble (bleomycin)
resistance gene contained in the Tn5 transposable element (eg
DH5aF´IQ, SURE, SURE2) will be resistant to Zeocin. Use E. coli
strains  that do not contain the Tn5 gene (eg TOP10,DH5, DH10, etc.).
To optimize selection in E. coli, the salt concentration must be < 110
mM and the pH must be 7.5. For more info see:

http://www.invitrogen.com/content/sfs/manuals/zeocin_man.pdf



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