Hi Amit,
Normally, we optimize annealing conditions with labeled DNA (cy5, FAM) -
adding MgCl2 and KCl usually improves annealing efficiency. You can
detect fractions of single and double stranded DNA on standard PAGE
gels. We then use the same condition to anneal the crystallization
reagents.
Hi Ronaldo,
We immediately desalt after His-Trap (desalting column connected in series)
into an Imidazol free and low salt buffer and digest with 1:50 to 1:100
TEV:substrate ratios (w:w) at RT or in the cold room (time often a compromise
between protein precipitation and completion of the cut)
).
Ralf
Ralf Jauch
Genome Institute of Singapore
60 Biopolis Street
#02-01Genome
Singapore 138672
Tel: (65) 6478 8102
Mobile: (65) 96398715
[EMAIL PROTECTED]
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Von: CCP4 bulletin board im Auftrag von mesters
Gesendet: Mi 13.02.2008 01:07
An: CCP4BB
