Hello Mates, I need to know about HEPES concentration to make E. Coli competent cells . Previously, I am using 0.1 M CaCl2, but now i want to use HEPES only, Thanks for suggestions.
Michael Delannoy
Muhammad,
The key here is what is the osmolarity of your cells? We usually aim for 300 mOsmols. but some organisms prefer lower (and higher I am sure). i.e. we found certain strains of E.coli prefer 20 mosmols. For your case here are some examples for HEPES:
50 mM Hepes with 3 mM MgCl2 and 3% sucrose = 150 mOsmols
100 mM HEPES with 3 mM MgCl2 and 3% sucrose = 223 mOsmols
100 mM HEPES with 5 mM MgCl2 and 3.5% sucrose = 243 mOsmols
These are used as TEM buffers.
good luck
Michael Delannoy
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