Team:Alberta/Notebook/protocols/sequencing

From 2010.igem.org

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==General Protocols==
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-----------------------------
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*[[Team:Alberta/Notebook/protocols/invitro_biobyte_assembly | In Vitro BioByte Assembly]]
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-----------------------------
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*[[Team:Alberta/Notebook/protocols/LB | LB Plates and Broth]]
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*[[Team:Alberta/Notebook/protocols/transformations |Transformations]]
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*[[Team:Alberta/Notebook/protocols/overnight |5mL Overnight ]]
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*[[Team:Alberta/Notebook/protocols/glycerol | Glycerol Stock ]]
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-----------------------------
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*[[Team:Alberta/Notebook/protocols/miniprep | Plasmid Miniprep ]]
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*[[Team:Alberta/Notebook/protocols/digest | Restriction Digest ]]
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*[[Team:Alberta/Notebook/protocols/vector_dephos | Vector Dephosphorylation]]
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*[[Team:Alberta/Notebook/protocols/ligation | Ligation ]]
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-----------------------------
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*[[Team:Alberta/Notebook/protocols/agarose_gel | Agarose Gel Electrophoresis ]]
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*[[Team:Alberta/Notebook/protocols/gel_extraction | Gel Extraction ]]
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-----------------------------
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*[[Team:Alberta/Notebook/protocols/pcr | PCR]]
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*[[Team:Alberta/Notebook/protocols/colony_pcr | Colony PCR ]]
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*[[Team:Alberta/Notebook/protocols/pcr_purification | PCR Purification ]]
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-----------------------------
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*[[Team:Alberta/Notebook/protocols/labelling | Sample Labelling Conventions]]
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*[[Team:Alberta/Notebook/protocols/sequencing | Fluorescent Sequencing Reaction]]
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*[[Team:Alberta/Notebook/protocols/primer_design | Primer Design]]
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-----------------------------
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Latest revision as of 02:31, 27 October 2010

TEAM ALBERTA


Fluorescent Sequencing Reaction

Procedure:

  • In a 0.2ml PCR tube, add:
Template 5.0ul (200 ng/ul)
VF primer 1.0ul
Dilute buffer 2.5ul
BD sequence mix 1.5ul
  • Mix well.
  • Select program 'seq-dye' on PTC 200 thermal cycler.
  • Run program. It will take about 2 hours.
  • Remove tube from PCR machine and transfer 10ul rxn mix to 1.5ml Eppendorf tube. Add:
Blue NaOAc/EDTA 1.5ul
95% ethanol 40ul
  • Let sit on ice 15 minutes.
  • Centrifuge 10 minutes at max speed.
    • Should see a small blue dot at bottom of tube.
  • Discard supernatant.
  • Wash pellet with 500ul 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 minutes.
  • Place in -20oC freezer to be delivered to MBSU 4th floor microbiology M534. Reactions delivered before 2pm will normally be returned the next day.


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