Cycle Sequencing Reactions for Single and Double-Stranded DNA

Notes:

1 reaction= 1 primer plus template (you will need 2 reactions if you have a forward and reverse primer).

Big Dye Sequencing Buffer 5x is available in the Core Lab if you purchased Big Dye from us.

Full Reaction (as recommended by ABI):

For each reaction add the following reagents to individual tubes:

1/2 Reaction:

For each reaction add the following reagents to individual tubes:

1/4 Reaction:

For each reaction add the following reagents to individual tubes:

After setting up your reactions:

Mix well and spin briefly. Run the cycle sequence reaction in a thermal cycler. Clean up the cycle sequence products to remove access Big Dye using Sephadex columns or another method. Dry samples down in tubes or a 96-well plate using a Speed Vac for 25-30 min. Add 15 ul formamide to each reaction and spin briefly to make sure there are no bubbles (if your samples were dried down in tubes, mix and make sure the liquid is all at the bottom of the tubes then let sit at room temp. for 2-10mins to let your sample go into solution. Mix briefly and then transfer to a 96-well plate). Denature at 94 C for 3 min. Rapid cool on ice ~5-10 min. Give to a Core Lab technician to load.