Team:IIT Madras/Protocols

From 2010.igem.org

PCR Protocol

Composition of PCR reaction:

• PHusion Phusion® High-Fidelity DNA Polymerase(from Finnzymes) - 0.2 mul
• 5x Phusion HF Buffer (from Finnzymes) - 4 mul
• dNTP – 1 mul
• DNA Template – 1 mul
• Primers(from Bioserve) – 1 mul each.
• Water – 12.8 mul

Total volume for the reaction is 20 mul.

Digestion Protocol

Composition of the digestion mixture:

• DNA - 4 mul
• Enzyme 1 - 1.2 mul
• Enzyme 2 - 1.2 mul
• Buffer - 2 mul
• Water - 11.6 mul

Total volume for the reaction is 20 mul.

Steps:

1. For EcorRI, PstI – The reaction mixture is kept at 37 degrees for 1.5 hours. For other Enzymes – The reaction mixture is kept at 37 degrees for 5 hours.
2. Inactivate enzyme by heating at 80 degrees for 20 mins.

T4 Ligation Protocol

Composition of Ligation mixture:

• T4 Ligase Buffer - 1mul
• 6:1 molar insert:vector (vector~10ng)
• miliQ water - (8.5 - DNA volume) mul
• T4 ligase – 0.5 mul

Steps:

1. Leave reaction at 22.5degC for 30min
2. Denaturate ligase at 65degC for 10min
3. Store at -20degC

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