Getting through PolyG Regions
? at aecom.yu.edu
Fri Apr 13 19:15:35 EST 2001
you can think of GC and G as being identical, since both elicit the same
degree of secondary structure.Also, there may be issues relating to
differential efficiency of base incorporation, but practically neither
has an impact on how you sequence clones : For both G-rich and GC-Rich
clones, try dGTP + O.5M betaine or 5% DMSO.
However, what appears to work even better is mixtures of big dye spiked
with dGTP : Try 4:1 of Big dye: dGTP initially but if that doesnt work,
optimise for your clones by performing an inverse titration of the two
big dye chemistries.
Maybe combine the mix with betaine or try Rx enhancer buffers from Gibco
if you can lay your hands on them : D and E have been particularly
efficacious ih our hands with regard to G-rich areas.
Finally, you could linearise the template as well; This will facilitate
melting of secondary structure, which is complicated in Closed covalent
circularised plassmids by virtue of topological constraints.
Feel free to contact me for further clarification,
Laurence S Hall
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