Cycle Sequencing Protocol

(3100 Capillary Sequencer)

Keep Dye Terminator Mixes on ice!

Wear gloves at the sequencing bench!

1. For each reaction mix the following reagents in a 0.2 ml tube. Label tubes with initials and numbers.

Reagent	Standard Protocols	LPGT Protocols
Big Dye Terminator Ready Reaction Mix (V2 or V3)	8.0ul	2.0 ul
Template Single Stranded DNA Double Stranded DNA PCR product DNA	50 - 100 ng 200 - 500 ng 1 - 100 ng ( depending on size)	300 - 450 ng
Primer	3.2 pmol	10 pmol
Deionized water	q.s.	q.s.
Total volume	20ul	8ul

Reagent

Quantity

Big Dye Terminator Ready Reaction Mix	16.0 ul	16.0 ul
Template Bacs and Cosmids Bacterial Genomic DNA	0.5 - 1.0 ug -	- 2 - 3 ug
Primer	5 - 10 pmol	6 - 13 pmol
Deionized water	q.s.	q.s.
Total volume	20ul	8ul

2. Mix well and spin briefly.

*It is recommended that cycle sequence reactions be optimized before running large numbers of samples.

Performing Cycle Sequencing

1. Run the samples using your thermal cycler or LPGT Thermal Cyclers. On PE 9600, two PE 9700 and one MJ Tetrad are available at LPGT. The running conditions are suggested as:

Standard

BAC and Cosmid

Bacterial genomic DNA

Place the tubes in a thermal cycler and set the volume to 20 µL.

40ul

Repeat the following for 25 cycles:

96°C for 10 seconds
(Denaturation)
50°C for 5 seconds(Annealing)
60°C for 4minutes(Extension)

Heat the tubes at 95°C for 5 mins.

30 cycles:

95°C for 30 seconds
50-55°C for 10 seconds
60°C for 4 minutes

Heat the tubes at 95°C for 5 mins.

45 cycles:

95°C for 30 seconds
50-55°C for 20 seconds
60°C for 4 minutes

Rapid thermal ramp to 4°C and hold until ready to purify.

Spin down the contents of the tubes in a microcentrifuge.

2.Proceed with Spin Column Protocol for tube users, and Isopropanol Protocol for 96 well plate users.

ABI Prism Dye Terminator Premix Contents:
A-Dye Terminator, C-Dye Terminator, G-Dye-Terminator, T-Dye Terminator,dITP, dATP, dCTP, dTTP,
Tris-HCl, MgCl 2, thermal stable pyrophosphates,
AmpliTaq DNA Polymerase, FS