Sequencing:

1. I start by generating high quality DNA. I normally do this by inserting my PCR products of choice into a plasmid vector, growing up a log phase culture, and harvesting the plasmids via a Promega Wizard Plasmid Mini-prep kit or equivalent (Qiagen works well too). This generally yields plenty of high quality DNA. I used to quantify the DNA using the 260/280nm method but I found that it was a waste of DNA and it was unnecessary because of the reproducibility of the mini-prep kits.

2. The sequencing PCR program comes directly from ABI -- I have not modified the program at all. The actual reaction, however, I have modified to fit out needs and to reduce the cost per reaction.

3. The PCR reaction has been cut by 75%. So, my typical reaction consists of 1ul of the high quality Plasmid DNA, 2ul of the ABI Big-Dye reaction mix (down from 8ul), and 2ul of primer (at a concentration of 1uM that equates to 2pmol of primer per reaction) for a total of 5ul per reaction. I sequence both strands of DNA by doing a forward and backward reaction for each plasmid. I have always gotten great results with this reduction (no further reagents are needed nor is the "dilution kit" from ABI needed).

Precipitation (this is a critical step):
Reagents: 95% Ethanol and 3M Sodium Acetate pH 5.3

1. The precipitation has been scaled down 75% as well and the procedure has been modified to work in the .2ul PCR tubes.

2. To each PCR tube, add .5ul of your 3M Sodium Acetate pH 5.3 and 12.5ul of your 95% ETOH.

3. ABI says to place this on ICE for 20 minutes. I find that since you are precipitating such a small volume it takes longer for the smallest pieces to precipitate. I ,when time allows, let the reactions precipitate overnight at -20 degrees C. I have also done the precipitation on ice in the -80 for 2 hours but generally the slower precipitation method is more reproducible with less ambiguity when reading the traces.

4. After the allotted time passes, I centrifuge the tubes in a benchtop microfuge at 4000 RPM for 20 minutes. I CAREFULLY remove the supernatant and wash the pellet with 175ul of 70% ETOH (Ice cold from the -20). I centrifuge the tubes at 4000 RPM for 6 minutes to ensure that the pellet is attached to the tube. I then remove the supernatant and move on to drying the reactions.

5. Drying can be accomplished in one of two ways : A speed vac or by air drying in the hood. I prefer the later method because it generally yields higher quality DNA and it's more reproducible than using the speed vac (the speed vac over dries the samples). In the summer, I often have no choice but to use the speed vac b/c of the humidity on Long Island -- it generally works fine but I still prefer the slower evaporation of the ETOH.

Protocol adapted from Kris's email.