High-throughput sequencing protocol

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.

Master mix

# of reactions-->

1
96 (100) 384 (400)

BDT
1µl 100µl 400µl

Primer @15pmol/µl
0.5µl 50µl 200µl

DMSO is not added to 1/8 reactions 0µl 0µl 0µl

5x Reaction buffer
1.7µl 170µl 680µl

Molecular biology grade diH₂O as needed to bring final reaction volume to 6µl (including template)

µl

µl

µl

Total volume of master mix

µl

µl

µl

Template (Do not add to master mix)
200-400ng per reaction

Final reaction volume
6µl

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:
96 C for 10 seconds
50 C for 5 seconds
60 C for 4 minutes
Hold at 4 C

Finish protocol within 24 hrs. If necessary store at 4ºC. (Your samples may get moved to a fridge if in JBPC cycler.)
Proceed to Isopropanol precipitation protocol.

Last updated 07/14/04

Master mix
# of reactions-->	1	96 (100)	384 (400)
BDT	1µl	100µl	400µl
Primer @15pmol/µl	0.5µl	50µl	200µl
DMSO is not added to 1/8 reactions	0µl	0µl	0µl
5x Reaction buffer	1.7µl	170µl	680µl
Molecular biology grade diH₂O as needed to bring final reaction volume to 6µl (including template)	µl	µl	µl
Total volume of master mix	µl	µl	µl
Template (Do not add to master mix)	200-400ng per reaction
Final reaction volume	6µl

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:	96 C for 10 seconds 50 C for 5 seconds 60 C for 4 minutes Hold at 4 C