From GenoSeq

Plasmid Cycle Sequencing Reaction

template	x1 µl	800 ng
BigDye Terminator Mix	2 µl
Sequencing Buffer	2 µl
primer (10 µM)	2 µl	20pmol
water	x2 µl
Total volume	10 µl

The above guidelines for the amount of BigDye per reaction are from the manufacturer.

In actual practice, 1 µl of BigDye is more than adequate for each reaction.

Adjust the volume with water.

Cycle Sequencing profiles for Plasmid

repeat the following for 25 cycles
96 °C	10 sec
50 °C	5 sec	(50 °C -58 °C depending on your primer's Tm)
60 °C	4 min
rapid thermal ramp to
4 °C

primers the core provides:

M13F primer: -21 M13F : TGT AAA ACG ACG GCC AGT

M13 R primer: -28 M13R : AGG AAA CAG CTA TGA CCA T

T7 primer: TAA TAC GAC TCA CTA TAG GG

T3 primer: ATT AAC CCT CAC TAA AGG GA

SP6 primer: ATT TAG GTG ACA CTA TAG

BGHR primer: TAG AAG GCA CAG TCG AGG