sequencing - too many N's in reaction (Jan/12/2009 )

Hi,

I'm having trouble with my sequencing protocol. Tgere are too may N's in the sequencing results. I'm using Big Dye (from ABI, cat# 4336917).

I'm making up a 10ul reaction
Big Dye 2 ul
5% buffer 2 ul
primer 1 ul (I've tried 10um and 3.2um concentrations)
Water 2 ul
Template 3 ul

A. PCR the recation mixture:
96°C 1min
25 cycles as following
96°C 10s
50°C 5s
60°C 4min
4°C forever

To the PCR reaction mixture add the following and transfer to a 1.5ml tube.
1. 125mM EDTA 5 ul
2. 95% ethanol 60 ul, vortex
3. Cover the tubes with aluminium
4. Precipitate -20°C for 30mins
5. Centrifuge at 13,000 rpm for 10min at 4°C, discard supernatant
6. Wash with 60 ul of 70% ethanol, vortex, centrifuge at 13,000 rpm for 10min at 4°C
7. Dry in speedrac for 10mins
8. Resuspend sample in 11ul HI-Di(injection buffer), by vortex
9. Denature at 95°C for 2 mins.

My 95% ethanol is not ice cold.

Any tips would be GREATLY appreciated.

Thanks

Do you know the "source" of your interference-ie. is is noise because of low signal strength or is there some other sequence that you are sequencing like another product that is making the sequence messy?