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From 2013.igem.org

Cloning Protocols Workflow

Workflow:

• 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