Protocol for BigDye Preparing sequencing samples: 1. Combine the following in a 0.2ml PCR tube: 250-500ng DNA/sample, 2.

2pmol primer/sample, add PCR water to total volume of 8ul.

2. Add 4ul per sample of Master Mix (see recipe below). Big Dye v3.1: 2 ul Big Dye dilution buffer 2 ul

3. Run the sample in a ThermalCycler using the BigDye protocol as follows: Step 1 Incubate at 96.0C for 20 seconds Step 2 Incubate at 50.0C for 20 seconds Step 3 Incubate at 60.0C for 4 minutes Step 4 Repeat to Step 1 for 39 times Step 5 Incubate at 12.0C forever Step 6 End

4. Clean up the reaction with various methods. Quantity of template DNA varies depending on the size of fragment. Template size: Quantity 100-200bp 500bp 1000bp 2000bp 3000bp

15-30ng 30-65ng 65-130ng 125-250ng 250-500ng

Big Dye dilution buffer: Tris-HCl,pH8.8 MgCl2 400mM 10mM

Collier Seq Protocol 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.

Cleaning with Sephadex Cleaning Big Dye reaction with Sephadex G-50 Materials: 1. Sephadex G-50 DNA grade (fine; available through many distributors including Pharmacia and Sigma. G-50 Med or Fine also works) 2. Columns (96-well or used AutoSeq G-50 columns from Amersham). 3. Autoclave 4. Centrifuge Method: 1. Determine the number of columns that will be used. 2. Weigh out the sephadex and put it in an autoclave safe glass bottle. (use 2 g. Sephadex G-50 with 80 ml. of water) 3. Autoclave the mixture for 15 minutes and store in a refrigerator. Note: Autoclaving will speed up the hydration of the sephadex and kill any fungus that may show up if the slurry is not used immediately. 4. Decant excess water after autoclaving so that there is approx. 1/3 water with 2/3 sephadex. 5. Transfer 750 ul of well shaken slurry to the top of each of the columns. Note: Make sure the slurry is thoroughly mixed and uniform before adding it to a column. If this is not done, the sephadex "pillars" may not be uniform and the results from the sample clean-up may not be good. 6. Spin down the G-50 column at 2000xg (5000 rpm in Eppendorf microcentrifuge) for 1 minute to remove the water (if using a standard microfuge with variable speeds a setting of 5 out of 14 works well). Spin the 96-well plates at 750xg for approximately 2 minutes. Note: The columns should be spun in a tube that it will collect the water from the hydrated sephadex. A waste tray should also be used for the 96-well tray. 7. Transfer the columns to either the sample collection tray (96-well) or a 1.5-ml labeled microfuge collection tubes. 8. Add the sample to the middle of the sephadex bed. 9. Spin the sample again at the same speed and time as in step 6. 10. Dry down the sample. 11. The remaining resin can be tapped out and the column re-used indefinitely after washing with tap water and rinsing with dH2O. For the 96 well columns, it may be necessary to spray out some of the chambers to remove all the used matrix.