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From 2013.igem.org

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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:

• Competent Cell Stocks
• LB and TB Media
• Pouring Agar Plates

Instruments:

• Nanodrop Protocol

See also:

• General Biotechnology Information
• SOS Lab Protocols
• http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf