Team:Alberta/Project/Sequencing

From 2009.igem.org

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<li>template 5.0 ul (200 ng/ul)
<li>template 5.0 ul (200 ng/ul)
           <li>VF primer 1.0 ul
           <li>VF primer 1.0 ul
-
           <li>dilute buffer 2.5 ul (said BD dilute buffer on tape of cap) in PCR/Sequencing box
+
           <li>dilute buffer 2.5 ul (reads BD dilute buffer on tape of cap) in PCR/Sequencing box
           <li>BD sequence mix 1.5 ul (said BD)  
           <li>BD sequence mix 1.5 ul (said BD)  
  </ul>
  </ul>

Revision as of 19:53, 21 October 2009

University of Alberta - BioBytes










































































































Sanger Sequencing Reaction

Procedure

  • In a 0.2 ml PCR tube add
    • template 5.0 ul (200 ng/ul)
    • VF primer 1.0 ul
    • dilute buffer 2.5 ul (reads BD dilute buffer on tape of cap) in PCR/Sequencing box
    • BD sequence mix 1.5 ul (said BD)
  • Mix well.
  • Select program 'seq-dye' on PTC 200 thermal cycler
  • Run program. It will take about 2 hrs.
  • Remove tube from PCR machine and transfer 10 ul rxn mix to 1.5 ml Eppendorf tube. Add
    • 1.5 ul Blue NaOAc/EDTA
    • 40 ul 95% ethanol
  • Let sit on ice 15 min
  • Centrifuge 10 min max speed
  • Should see a small blue dot at bottom of tube
  • Discard supernatant
  • Wash pellet with 500 ul of 70% ethanol
  • discard supernatant and spin briefly to bring down the residual liquid. Draw liquid off with a P10 pipette tip. *Do not disturb the pellet.*
  • Air dry for 10 min
  • Place in -20 C freezer to be delivered to MBSU 4th floor microbiology M534. Rxns delivered before 2pm will normally be returned the next day.

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