**BigDye has much stronger signal if your reactions are ok using regular chemistry, you can cut at least to ½, probably ¼ immediately. Beyond this point, it is best to determine empirically whether your reactions will work.**
 
10 uL reaction
4, 3, 2, or 1 uL of BigDye reaction mix
0,1,2, or 3 uL of 2.5X Dilution Buffer
10 pmol primer
250-500 ng of plasmid (@3-5kb) target
dH20 to 10 uL
 
20 uL reaction
8, 6, 4, or 2 uL of BigDye reaction mix
0, 2, 4, or 6 uL of 2.5X Dilution Buffer
20 pmol primer
250-500 ng of plasmid (@3-5kb) target
dH20 to 20 uL
 
Cycle Conditions:
95, 15 seconds
50, 15 seconds
60, 2:30 min (can cut down if you are in a hurry)
25 cycles
Dilution Buffer (2.5X) 20$/ mL from PE-Cetus
200 mM TrisHCl
5 mM MgCl2
pH to 8.3
Loading Buffer (5:1) Formamide:EDTA
Deionized Formamide (MolBio Grade)
25 mM EDTA, pH 8.0 containing 50 mg/mL of dextran blue
 
Precipitation protocol for reactions (EtOH or isopropanol)
**Get 100% reagent grade EtOH or isopropanol**
**open bottle and dilute to 75% with dH20**
this is because the 100% reagents will hydrate over time and the opening of the bottle, and you want to be 60% +/- 3% Final
 
add 40 uL (10 uL reaction) or 80 uL (20 uL reaction)
set at room temp for 15 minutes
spin at max at room temp for 15 minutes
aspirate pellet
air dry (do not overdry) for 1 minute
add 4 uL of loading buffer above