Sequencing with deaza-dGTP and signal loss

Bill Nowatzke wnowatzk at ucs.indiana.edu
Wed Mar 16 15:57:19 EST 1994


I am new to sequencing and have been working out conditions to sequence a
gene cloned in a Bluescript plasmid. The gene has a 75% G+C content, so I
am using a-35S-dCTP as the label and replacing dGTP in the label mix with
deaza-dGTP. The main problem that I have been encountering is that my 'C'
lane is only readable about 1/3 of the way up the gel and fades to a
clear lane with no sequence? while the other three lanes still have
bands. I am preparing my template DNA by M13 ssDNA procedures. After
annealing the primer I slow cool to RT, and then add label mix ( 1 uL
35S-dCTP (about 10 uCi/RXN, sp act is 1 Ci/mmol), dNTP's = dATP, dTTP,
deaza-dGTP to final conc 1.5 uM, DTT, H2O, and Sequenace. RT for 2 min, I
then terminate with ddNTP's 10 min at 37C, stop with formamide+dye. I did
not prepare the ddNTP's and am wondering if it is an absolute requirement
to use deaza-dGTP (and if the conc must be about 2X rather than if dGTP
were used) or if I can just use ddGTP? I am also wondering if a small
amount of unlabelled dCTP should be included in the labelling mix? Are
there any special consideration for the ddNTP's when using deazaG? Any
advise would be greatly appreciated. Bill Nowatzke.



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