Cycle sequencing anyone?

Ed Marsden ed.marsden at utoronto.ca
Sat Mar 22 22:51:13 EST 1997


Netters,

For the sake of simplicity, I've attempted to cycle sequence both the 
commercial plasmids and my own ds isolate as opposed to using the 
standard protocol (we're using the Boehringer Taq polymerase kit and DIG 
detection).

	Here's the scoop: After X-ray development, we cannot see ANY 
bands - only background. The first x-ray was pretty dark so we cut the 
exposure time in half which gave us less blackened background but still 
no ladder (except mabye the wells?)
	My professor and I agree that the problem may be due to the 
sequencing reactions (I've followed the Boehringer protocol 
religiously for all steps). I'm wondering if it would be advisable to 
boost the concentrations of template, buffer, primer et al. and add more 
dH2O to reduce the viscosity of the mixes? Increase the number of 
cycling steps? 
   I don't believe that the detection protocol is awry as we do get 
residual background due to the chemiluminescence (blotches on the film 
due to a poor blocking job). The nylon transfer seems to be working well 
also...
	Any suggestions would be appreciated.
							-Neil



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