Disclaimer: While this protocol has worked very well for the Bay Paul Center, we must stress that ABI will not support any sequencing protocols other than those written in their documentation (1X).
Reactions should be cleaned up (by precipitation) within 24 hours. After precipitation they should be sequenced within 3 days. If longer times elapse your sequences will be somewhat shorter. If long reads are a priority, be sure to contact JBPC to schedule your sequencing before you set up reactions.
Template is the most important variable in your sequencing. Too much or too little is likely to produce short or faint reads respectively. Poor quality template can produce a number of possible aberrations. If you are uncertain or having trouble, contact JBPC or ABI tech support for help.
Supplies: BDT, primer (5-15 pmol/µl), 5X Rxn Buffer*, deionized water, ABI 96 or 384 well reaction plate, plate cover suitable for thermocycling
Equipment: pipette (8 or 12-channel preferred), centrifuge, thermocycler
*Note: 5 X Rxn Buffer = 400mM Tris-HCl, pH9.0 containing 10mM MgCl2 (keep refrigerated).
1) Defrost BDT on ice. |
2) Spin template plate briefly to pool samples if necessary. |
3) Make master mix (see below) and keep on ice. |
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# of reactions-->
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1
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96 (100) | 384 (400) |
BDT
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1µl | 100µl | 400µl |
Primer @15pmol/µl
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0.5µl | 50µl | 200µl |
DMSO is not added to 1/8 reactions | 0µl | 0µl | 0µl |
5x Reaction buffer
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1.7µl | 170µl | 680µl |
Molecular biology grade diH2O as needed to bring final reaction volume to 6µl (including template)
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µl
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µl
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µl
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Total volume of master mix
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µl
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µl
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µl
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Template (Do not add to master mix)
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Final reaction volume
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4) Aliquot Mastermix to each well of reaction plate. Actual amount will depend on how much diH2O you have added. | |
5) Add 200-400 ng of template to each well of reaction plate. | |
6) Cover 96-well plate with cover mat or strip tubes; 384 well plate with cycle seal. | |
7) Quick spin to settle plate contents. | |
8) Thermocycle (program "BIGDYE" if using JBPC cycler) | |
Repeat 25 cycles:
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96 C for 10 seconds 50 C for 5 seconds 60 C for 4 minutes Hold at 4 C |
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