The following manipulations are what we do for sequencing DNA. These conditions work well greater than 90% of the time. When they don’t work, it’s usually a problem in the template or primer that was provided.
DNA (ds = 100-200 ng for < 20 kb plasmid, PCR = 10ng/100 bp, lg. plasmids, BACs, cosmids 500 ng-1 ug)
Primer (5-20 pmol; for PCR fragments stick with 5)
Enzyme Reaction Mix: 2 ul BigDye plus 3 ul buffer (extra buffer can be purchased from Applied Biosystems: Part Number 4336697, 1 ml for $25)
1 ul DMSO (optional) then water to 20 ul.
Very similar results are obtained from the following different cycling conditions:
96° 3', then 35 cycles of 96° 10" 50° 15" 60° 3', finish with 72° 7'
96° 3', then 35 cycles of 96° 10" 55° 15" 60° 3', finish with 72° 7'
96° 3' for "hot start", then 50 cycles of 96° 10", 58° 4’, finish with 72° 7'.
Secondary structure:
Supplement reaction with betaine to 1M final, enzyme mix is a cocktail of dGTP BigDye:normal BigDye at 1:3 or 2:2. Do extension at 68 or 72°
Cleanup:
Add 10 ul CleanSeq beads
Add 80 ul 80% EtOH (for DYEnamic ET and fluorescent fragments use 100%)
Mix by pipetting
Put on magnetic plate 2-3’
Withdraw liquid
Add 200 ul 80% EtOH
Withdraw as much liquid as possible
Remove from magnetic plate (further drying not required)
Add back 40-50 ul water
When beads have fully released from the side, transfer 10 ul to plate for capillary sequencing
Here is additional info on how to order the beads from Agencourt Bioscience:
CleanSEQ starter kit P/N 000145 $??? - includes the magnetic plate and 8 mls of beads
CleanSEQ 8 ml reagent P/N 000121 $408
CleanSEQ 50 ml reagent P/N 000136 $2125
Orders at 1-800-361-7780 Reference the quote # AGEN 7050 (# may change, talk to us if they give you problems)