Mix for sequencing - Big Dye buffer necessary or not? (Mar/02/2007 )
Hello Dear All,
I would like to perform a sequencing reaction with the Big Dye terminator 3.1 (ABI) on plasmid DNA but we have too much protocols in our lab ... Some of it preconize the utilisation of the big dye buffer and the others not!!!
Please help me I've too much reactions to make a mistake!!
I certainly recommend using the big dye buffer. It makes the probability of a successful sequencing run better.
My lab started without using the buffer. Succes rate was poor. When we started using the buffer, succes rates improved.
I have one comment thought. Don't use too much big dye. A problem that was plaguing my lab until recently was found to be caused by excessive use of big dye in our reactions. Use 1ul to 0.5ul per 10ul reaction.
You could check the big dye product sheet, it will give the protocol which should work. But if you use even 1/4 of the recommended volume, it works.
the buffer is there so you can use less DyeT, which is very expensive. I have used both 4uL DyeT (no buffer) and 2uL of DyeT (1uL buffer) per 10uL reaction and it always works well. But, you may need to let whoever runs the sequencing gel know how much you have used so they know how much to load on the gel.
I use -
300-400ng plasmid template
2 uL DyeT v3.1
1 uL buffer
1 uL primer (3.2 pmol per uL concentration)
water to a final volume of 10uL.
I'm sure others have slightly different protocols, but I have found this to work well.