seq problems

hauge at Frodo.MGH.Harvard.EDU hauge at Frodo.MGH.Harvard.EDU
Thu Dec 15 06:28:56 EST 1994



We are sequencing a clone which contains a region of roughly 300 bp which 
contains about 80% C (or G depending on which strand). We are 
sequencing single strand templates using sequenase. The sequence prior to
the C rich region looks great, then we have a weak stop (bands in all 4 
lanes) In the C tract the sequence looks great and continues for several
100 nucleotides. However, in the G,A and T tracts, the intensity of the 
bands decreases by about 2 orders of magnitude. So in other words, C lane 
looks great but the others basically stop. Changing the ratio of dideoxys
has no effect. If we vary the time of the labeling rxn (3', 5', 10' 15' 
and 20') we do see an effect. At 3' and 5' C lane looks good and G, A, T 
reactions just stop. At 10', 15' 20' the G lane looks good extending 
beyond the previous stop giving nice sharp bands, but now the A, T,C 
reactions have stopped (intensity down 2 orders of magnitude). The 
exception is at 15' where we begin to get clear sequence in the A lanes.
In other words, at 15' labeling we get sequence in the G, A lanes but
nothing in the T and C lanes. 
Any expaination for what could be going on?
    				Thanks Brian Hauge

 



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