Big Dye Protocols and Notes - Plasmid Templates

Updated September 11, 1998

Hi all,

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,

--broe

++++++++++++++ Big-Dye Protocols and Notes +++++++++++++++

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.

============ For Plasmids =============

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 1:2 diluted ABI Stock*	10-30	200-400	5-7 ul	25 or 45
1/12 reaction	2 ul 1:3 diluted ABI Stock**	10-30	200-400	5-7 ul	45 or 60
1/16 reaction	2 ul 1:4 diluted ABI Stock***	10-30	200-400	5-7 ul	80 or 99
1/20 reaction +++++++++	2 ul 1:5 diluted ABI Stock****	10-30	200-400	5-7 ul	99 (see Note #6)

    *1:2 diluted ABI stock = Dilute 2 ul of ABI stock with 2 ul of 5x rxn buffer
    **1:3 diluted ABI stock = Dilute 2 ul of ABI stock with 4 ul of 5x rxn buffer
    ***1:4 diluted ABI stock = Dilute 2 ul of ABI stock with 6 ul of 5x rxn buffer
    ****1:5 diluted ABI stock = Dilute 2 ul of ABI stock with 8 ul of 5x rxn buffer

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:

Rapid thermal ramp to 95degC
95degC for 10 sec.
Rapid thermal ramp to 50degC
50degC for 5 sec.
Rapid thermal ramp to 60degC
60degC for 4 min.
followed by rapid thermal ramp to 4degC and hold until ready to purify through G-50 microtiter plate spin columns.

Remember, Taq polymerase is thermo-stable and several in my group have shown that the more cycles, the better the signal using the FS thermo-cycling conditions. So if you are going to do the cycling overnight, might as well use 99 cycles and get stronger signals!!

Note #6: The Modified Cosmid, BAC, PAC, Fosmid Thermo-cycling Conditions **Also Improves the Read Lengths for Plasmids with the Big-Dyes**

95degC hold for 5 min. (ONLY needed if Thermofidelase is used but do the hold anyway and only use Thermofidelase as a last resort.
Then, with 18-22mer primers use 99 cycles of the following:

Rapid thermal ramp to 95degC
95degC for 30 sec.
Rapid thermal ramp to 50degC
50degC for 20 sec.
Rapid thermal ramp to 60degC
60degC for 4 min.
followed by rapid thermal ramp to 4degC and hold until ready to purify through G-50 microtiter plate spin columns.

Additional NOTES:

There have been some posting of questions to autoseq@net.bio.net and here is a copy of a reply I sent to a question about some difficulties with ds template preparations and other strangeness.
A couple of things to suggest, look at the protocol we are using for the ds template isolations. Essentially it's a quick and dirty (actually rather clean) cleared lysate *without* all the extra stuff like Wizzard or filtering or phenol.
Second is to do a template concentration study with the Big dye reactions. Quite supprisingly, we and others have observed that 2 to 5 times *less* template than was used previously with the dRhodamines seems to give much better results in most instances. Strange but rather true.
Third, for regions that are very GC rich, it seems that going back to the dRhodamines often reads through them when the Big Dyes fail. Oh BTY, we always use either 5% or 10% DMSO in both the dRhodamine and BigDye reactions and are use the dRhodamines at 1/4 reaction (5-7 ul total reaction volume) and the Big Dyes at 1/16 reaction (also a 5-7 ul total reaction volume).
Last but not least, it seems very clear from lots of expts. that EDTA inhibits the Taq Polymerase. Thus, instead of dissolving our template DNA in TE or diluted TE, we now routinely are just using sterile ddH2O. I'd much rather keep the EDTA there to inhibit nucleases but its bad effect is severe enough that steril ddH2O is a better choice.

Bruce Roe, broe@ou.edu