question about big dye..please help

[molbio123]

I use big dye in my sequencing reactions. I use it in a reaction, place the reaction on the thermocycler. The reaction ends at 4 deg C Hold on the thermocycler.

I keep big dye at -20 deg C frozen. But how long can I keep the sequencing reaction at 4 deg C Hold? Would it still be ok to sequence if left at 4 deg after a weekend or when does it go bad? thanks.

[Tattersail]

According description you can store it at 4°C. So shouldn't be a bigger problem to let it in the thermocycler at 4°C over the weekend.

Though I never used big dye, but you can still check the description manual to your kit, can't you?

Problematic is usually just continuous freezing and rethawing. But that applies to most of chemicals and reagents.

[danfive]

4 C in the dark---couple of days should be fine.
Everything still depends on how good your initial template was.
If its something easy your sequence should like fine.
If its already a tough sequence---then it will probably get worse.

[jiajia1987]

I have had a friend who did her sequencing with Bigdye and holding it in the PCR machine at 4deg, and storing it in the -20deg freezer after that because she wanted to go home. The samples were stored over the weekend and it turned out fine for her. her gene is 0.3kb. when I store my samples for too long, it can get a little messy. my gene is 1.5kb.

[spal]

When I use BigDye, I typically dry my samples with a vacuum centrifuge as soon as the cycle sequencing reactions are completed and the samples are cleaned. When dried, I can store them at room temperature for up to a week, if not more. I've had great success if I re-suspend the DNA in formamide immediately before sequencing.

I do on occasion leave the samples at 4 degrees for a day or two, and have not noticed a significant change in sequence quality. However, by drying the samples you can bypass the detrimental freezing/thawing that the previous poster referred to.