Back in 1998, we tested various reaction conditions for the ABI Big-Dye Terminators with double-stranded DNA templates ranging from pUC or pGEM without any insert up to bacterial genomes in the 2 Mbp size. Below are the results of these experiments and the conclusions we have reached. PLEASE BE ADVISED that any deviation from the ABI protocols as published in ABI's literature is not supported by ABI. I only am providing the information below so you will have an idea of what we have done but and the disclaimer is "use these protocols at your own risk". If you like them and are happy you learned about them, send me some e-mail. If they don't work for you, keep it to yourself... :-)
If you have any suggested changes to the protocols below, please let me know what experiments you have done and the results, so that I can implement them.
Thanks and happy sequencing,
Below are the sequencing conditions we have tested for the ABI Big-Dye terminators (PE-ABI #4303150 for the 1000 reaction kit - Description: TF,KIT BTD RR-1000) with various templates. As usual, it always is important to quantitate all templates by agarose gel electrophoresis vs size and concentration standards and do a few tests with different template concentrations to determine the optimal conditions for your reactions.
Although several conditions are given below, the ones we routinely use are underlined with ++++++'s. Notice also that you SHOULD NOT REDUCE either the absolute amount of primer or template when the reactions are scaled down.
|Reaction "size"||Big Dye Mix (ul)||Primer (pmoles)||Template (ngrams)||H2O to final volume of||Number of Cycles|
|ABI's 1x reaction||8 ul||10-30||200-400||20 ul||25|
|1/2 reaction||4 ul||10-30||200-400||10 ul||25|
|1/4 reaction||2 ul||10-30||200-400||5-7 ul||25|
|1/8 reaction||2 ul
|10-30||200-400||5-7 ul||25 or 45|
|1/12 reaction||2 ul
|10-30||200-400||5-7 ul||45 or 60|
|1/16 reaction||2 ul
|10-30||200-400||5-7 ul||80 or 99|
|10-30||200-400||5-7 ul||99 (see Note #6)|
Note #1: 5x rxn buffer = 400mMTris-HCl, pH9.0 containing 10mM MgCl2
Note #2: BOTH the 5x rxn buffer and the diluted ABI stock should be KEPT ON ICE and prior to placing the microtiter plate in the thermocycler, we seal them using Sealing Mats such as those available from Thermo Scientific or Corning.
Note #3: The 1/4, 1/8, and 1/12 reactions give very good results with the FS thermo-cycling conditions (Note #5), but the 1/12, 1/16 and 1/20 reactions give better results by using the Modified Cosmid, BAC, PAC, Fosmid Thermo-cycling Conditions without Thermofidelase (Note #6).
Note #4: All of the above reactions work with 5% or 10% DMSO.
Note #5: FS thermo-cycling conditions are as recommended by ABI for use with 18-22mer primers, but use 25, 35, 45, 60, 80 or even 99 cycles of:
Note #6: The Modified Cosmid, BAC, PAC, Fosmid Thermo-cycling Conditions **Also Improves the Read Lengths for Plasmids with the Big-Dyes**
Bruce Roe, firstname.lastname@example.org