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Team:IIT Madras/Protocols
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| - | + | <b>PCR Protocol</b> | |
| + | |||
| + | 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. | ||
| + | |||
| + | |||
| + | |||
| + | <b>Digestion Protocol</b> | ||
| + | |||
| + | 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: | ||
| + | #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. | ||
| + | #Inactivate enzyme by heating at 80 degrees for 20 mins. | ||
| + | |||
| + | |||
| + | <b>T4 Ligation Protocol</b> | ||
| + | |||
| + | 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: | ||
| + | #Leave reaction at 22.5degC for 30min | ||
| + | #Denaturate ligase at 65degC for 10min | ||
| + | #Store at -20degC | ||
| + | |||
| + | |||
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Revision as of 18:28, 27 October 2010
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:
- 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.
- 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:
- Leave reaction at 22.5degC for 30min
- Denaturate ligase at 65degC for 10min
- Store at -20degC
