ABI BigDye protocol
Note:
this is for 10 uL reactions (i.e. half-volume reactions) On
ice prepare in thin-walled 0.5 mL tubes:
- 1.6 pmol of
single primer (0.8 uL of 2 uM primer stock)
- Template DNA
15-45 ng (up to 5.2 uL).
- 4 uL BigDye
RR mix ("pink juice")
- Sterile D.D.
water, to 10 uL total.
- Mix and spin
down. Keep on ice.
or try the cheaper
option:
3-5ul
of clean PCR product from SAP reaction
1.6ul of 2uM primer
1.0ul Pink Juice
bring final volume to 10ul with water
Preheat thermocycler to 95oC by pausing at beginning of cycle.
Load samples.
Run as follows:
- 95oC
3 min
- 95oC
10 sec
- 50oC
5 sec
- 60oC
for 4 min
- Go to step
#2 (98 times)
- Soak cycle
at 4oC