Facility Sequencing Procedures

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 $55)

1 ul DMSO (optional) then water to 20 ul.

99% of in house reactions are cycled with the following protocol:

96° 2', then 35 cycles of 96° 10" 52° 15" 60° 3', finish with 72° 1'

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 to 20 ul reaction (you can use less with smaller reactions)
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 130 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 Beckman Coulter(Agencourt Bioscience) or Axygen (through your favorite distributor):

Beckman(Agencourt)

CleanSEQ starter kit - P/N A29156: $675.75 - includes the magnetic plate and 8 mls of beads
CleanSEQ 8 ml reagent - P/N A29151: $449.65
CleanSEQ 50 ml reagent - P/N A29154: $2237.50

Orders at 1-800-361-7780  Reference the quote # 10-5916 (# may change, talk to us if they give you problems)

Axygen(through your favorite distributor):

Magnetic Dye Clean 5 ml reagent - P/N MAG-DYECL-5   
Magnetic Dye Clean 50 ml reagent - P/N MAG-DYECL-50