377 protocols (last updated 2001-06-29)

The following is a list of methods currently being used to sequence customer samples. Methods include set up of sequencing reactions, thermal cycling conditions, and sephadex cleanup.

1. Sequencing reactions
1. Plasmid sequencing: You will need approximately 500ng of sample (the least you can get away with is 200ng)
1. Add 1.6 - 3.2 pmoles of primer (our concentrated stocks are at 320pmoles/µl, use a working solution of 3.2pmoles/µl and add1µl)
2. Add 500ng of sample
3. Add 1µl of ABI Big Dye vs 2.0 or 3.0 terminator kit.
4. Add 2µl of 5x Reaction Buffer
5. Adjust volume to 10µl with DIUF water.
6. Quick spin and cover with a drop of mineral oil and cap the tube.
2. PCR templates: You will need 10ng per 100 base pairs of the pcr product (e.g. if the pcr product is 1000bp you will need 100ng)
1. Add 1.6-3.2pmoles of primer (our concentrated stocks are at 320pmoles/µl, use a working solution of 3.2pmoles/µl and add1µl)
2. Add 10ng/100bps of sample
3. Add 1µl of ABI Big Dye vs 2.0 or 3.0 terminator kit.
4. Add 2µl of 5xReaction Buffer.
5. Adjust volume to 10µl with DIUF water.
6. Quick spin, then cover with a drop of mineral oil and cap the tube.
3. BAC or Phage templates: You will need approximately 4µg of DNA
1. Add 3.2-6.4pmoles of primer
2. Add 4µg of DNA
3. Add 8µl of Big Dye terminator kit
4. Adjust volume to 20µl with DIUF water
5. Quick spin, then cover with a drop of mineral oil and cap the tube
2. Thermocycler conditions
1. For plasmid and PCR templates
1. 96ºC for 2 min, then 60 cycles of:
1. 96ºC for 30 sec
2. 50ºC for 30 sec
3. 60ºC for 4 min
2. Followed by 4ºC hold
2. For BAC or Phage templates
1. 96ºC for 2 min, then 60 cycles of:
1. 96ºC for 45 sec
2. 50ºC for 45 sec
3. 60ºC for 4 min
2. Followed by 4ºC hold

If the template concentration is between 250 and 300ng/µl increase the number of cycles by 5 or 10.

If the template has a GC content greater than 70% increase the denaturation temperature from 96 to 98.

If the primer is long or GC rich you may have to increase the annealing temperature from 50ºC (calculate Tm= 4(G+C)+2(A+T), then drop the temperature 2-5ºC).

3. Sephadex clean up of the sequencing reactions
1. Preparing a Sephadex plate
1. Tape a V bottomed plate to the bottom of a Millipore filter plate
2. Fill the wells of the Millipore filter plate with 250µl of Sephadex
3. Centrifuge at 1500rpm for 3 min, empty the liquid from the v bottom plate and re-attach to the filter plate
4. Add another 250µl of Sephadex to the wells of the filter plate
5. Centrifuge at 1500rpm for 3min; replace the bottom plate with a clean v bottom plate
2. Clean up of samples
1. Transfer the sequencing reaction to the surface of the sephadex plug
2. Centrifuge at 1500rpm for 5min
3. Remove the v bottom plate and transfer it to a pre-warmed vacuum oven (70ºC)
4. Apply vacuum and leave to dry (this usually takes 30-45min)

If you are not using the samples immediately, cover the plate with the Thermoseal film and store at -20ºC. Samples can be stored for at least a week like this. Resuspend in 0.8µl of loading dye just before use.