Sanger sequencing - user prep samples

The BRF uses an AB 3730 capillary sequencer.  We recommend the protocols set out below.  Please ensure the follow the appropriate precipitation protocol for either 96-well reaction plates OR microfuge tubes.

Big Dye terminator 1 µL
Primer 3.2 pmol
5x sequencing buffer 3.5 µL
Template as required (see ABI protocols)
Milli Q water To 20 µL

Mix well and spin briefly.

Cycling conditions:

  1. 94°C for 5 minutes.
  2. 96°C for 10 seconds.
  3. 50°C for 5 seconds.
  4. 60°C for 4 minutes.

Repeat steps two to four 30 times, then hold at 4°C.

Purification of extension products

This is a critical stage.  Incorrect concentration of ethanol can lead to loss of short products and incomplete precipitation of DNA fragments.  Always make fresh 70% ethanol for the final wash step.  Poor cleanups that fail to remove all unincorporated dye terminators can lead to dye blobs in the results.  Excess salt in samples can interfere with the electrokinetic injection of the sample.

Ethanol/EDTA Precipitation (recommended protocol)

With the Big Dye terminator v3.1, the ethanol/EDTA precipitation method produces consistent signal, while minimizing unincorpirated dyes.  It is particularly good at removing unincorporated dye-labelled terminators.

NOTE:  while this method produces the cleanest signal, it may cause loss of small molecular weight fragments.IMPORTANT:  95% ethanol is usable, but you must make sure the final ethanol concentration for precipitation remains the same (67-71%).

To precipitate 20µL sequencing reactions in 96-well reaction plates

  1. Remove the 96-well reaction plate from the thermal cycler and briefly spin.
  2. Add 5µL of 125mM EDTA to each well.  Make sure the EDTA reaches the bottom of the wells.
  3. Add 60µL of 100% ethanol to each well.
  4. Seal the plate with aluminium tape and mix by inverting four times.
  5. Incubate at room temperature for 15 minutes.  Important:  proceed to the next step immediately.  If this is not possible, spin the plate for an additional two minutes before performing the next step.
  6. If you are using the Beckman Alegra 6A centrifuge with a GH-3.8A rotor, spin the plate at 1650g for 45 minutes.  For any other centrifuge use a plate adaptor and spin the plate at the maximum speed as follows:  1400-2000g for 45 minutes, or 2000-3000g for 30 minutes.
  7. Invert the plate and spin for up to 185g for one minute, then remove from centrifuge.
  8. Add 60µL of 70% ethanol (freshly made) to each well.  Spin at 1650g at room temperature for 15 minutes.
  9. Invert the plate and spin up to 185g for one minute, then remove from the centrifuge.  Note:  start timing when the rotor starts moving.  Important:  Make sure the wells are dry.  You may use a Speed-Vac for 15 minutes to dry the plate.  Important:  make sure the samples are protected from light while they are drying.  Store at -20°C.

To precipitate 20µL sequencing reactions in microfuge tubes

  1. Remove tubes from the thermal cycler, briefly spin and transfer products to 1.5mL microfuge tubes.
  2. Add 5µL of 125mM EDTA to each well.  Note:  make sure the EDTA reaches the bottom of the wells.
  3. Add 60µL of 100% ethanol to each well.
  4. Vortex and spin briefly.
  5. Incubate at room temperature for 15 minutes.  Important:  proceed to the next step immediately.  If this is not possible, spin the plate for an additional two minutes before performing the next step.
  6. Spin in a benchtop centrifuge at maximum speed (12,000-13,000 rpm) at room temperature for 20 minutes.
  7. Aspirate teh supernatant carefully.
  8. Add 250µL of 70% ethanol (freshly made) to each tube.  Spin at maximum speed in benchtop centrifuge for five minutes.
  9. Aspirate teh supernatant carefully.
  10. Dry pellets in Speed-Vac for 15 minutes.  Important:  make sure the wells are dry and the samples are protected from light while they are drying.  Store samples at -20°C.
Service: 
Sanger sequencing
Illumina technologies
Ion Torrent

Updated:  22 October 2017/Responsible Officer:  Director, ACRF/Page Contact:  Web Admin, ACRF