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RESEARCH DIVISION Laboratory Manual

 
 

Dye Terminator Cycle Sequencing

STORE REACTION PREMIX AT -20°C (O’CONNELL LAB).

THAW AND MAINTAIN ON ICE PRIOR TO USE

Add the following to a PCR tube:

1µg dsDNA
8µl BDT reaction premix
5 picomol primer
x µl dH2O

All reactions in 20µl

Overlay mix with oil

PCR REACTION:
- 96°C 30 seconds
- 50°C 15 seconds
- 60°C 4 minutes

25 cycles in total

(Program No.18- Bowtell lab PCR Machine)

 

Removal of Dye-Labelled Nucleotides Following Cycle Sequencing

Use Microspin G-50 columns.

Column Preparation:

  1. Resuspend the resin in the column by vortexing gently
  2. Loosen the cap and snap off the bottom closure
  3. Place the column in a 1.5ml screw cap microcentrifuge tube for support. Alternatively, cut the cap from a flip-top tube and use this for support.
  4. Pre-spin the column for 1 minute at 2000 x g (4600 rpm in Biofuge 13).

Sample Application:

  1. Place the column in a new 1.5ml tube and slowly apply the sample to the centre of the angled
  2. surface of the resin bed, being careful not to disturb the resin. Careful application of the sample to the centre of the bed is essential for good separation. Do not allow any of the sample to flow around the sides of the bed.
  3. Spin the column for 1 minute at 2000 x g. The purified sample is collected in the bottom of the support tube.
  4. Discard the column
  5. Dry the sample in a speedivac for at least 30 minutes
  6. Samples may now be sent to Angela Higgins for sequence analysis
Angela Higgins
Automated DNA Analysis Facility
School of Biochemistry and Molecular Biology
University of New South Wales Sydney 2052
Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998