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Team:Washington/Protocols
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Revision as of 07:28, 7 September 2013
==Cloning Protocols Workflow==
Tips and Etiquette:
- General Basic Protocol Information
- How to Label EVERYTHING
- Keep notebooks up-to-date
Workflow:
- Primer Design
- Streaking a Plate for Isolation
- Overnight Cultures
- 1) Isolation of Plasmid DNA (miniprep)
- 2) General PCR Protocol (also see PCR GoTag - product (30-200ng/ ul) Check on gel PCR Purification and/or Dpnl Digest
- 3) General Digestion Protocol Check on gel PCR Purification or Heat Inactivation (check enzyme temp and time, usually 80C for 20min)
- 4) Agarose Gel Electrophoresis
- 5) General Ligation Protocol (Don’t forget background control plates) Heat Inactivation (optional - up to 10 fold increase) - 65° for 10 minutes
- 6) Heat shock/chemical competent transformation
- 7) Colony PCR with Green taq Miniprep (stocks can be made from this culture - add 1ml extra)
- 8) DNA Sequencing
- 9) Making Glycerol Frozen Stocks
Media Preparation:
Instruments:
See also:
- General Biotechnology Information
- SOS Lab Protocols
- http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf
