Incompetent DH10B's?

John Dixon jpcd100 at mole.bio.cam.ac.uk
Tue Nov 23 10:33:46 EST 1999


Hi all, I have been using competent cells prepared by the Inoue
protocol for some years now, mainly XL1-blues and XL10-golds, with no
problems and competencies of >10E8. But recently I have needed to
switch to a strain with no antibiotic resistance markers already
present, (ie no Tet) and tried DH10Bs. Initially, using the standard
CaCl2 method in Maniatis I was getting up to 10E7, but when I tried to
prepare a storable batch using the Inoue method I got less than 10E6
twice running.

Is there a trick for DH10Bs, a different optimal harvest OD from 0.6
perhaps? Or is there a good way to store the CaCl prepared cells
(glycogen addition failed completely)?

Thanks for any tips

JD

-- 
John Dixon                    Lab 44 (1223) 334131
Wellcome/CRC Institute        Fax 44 (1223) 334089
Cambridge University
United Kingdom CB2 1QR       e-m: jpcd100 at mole.bio.cam.ac.uk




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