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Team:Stockholm/18 October 2010
From 2010.igem.org
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*#0.5 μl pEX.hS.RyC | *#0.5 μl pEX.hS.RyC | ||
| - | ===PCR verification for Uppsala team=== | + | ===PCR verification for Uppsala-Sweden team=== |
Helping the Uppsala team with a PCR verification of one of their assemblies. | Helping the Uppsala team with a PCR verification of one of their assemblies. | ||
Revision as of 18:21, 18 October 2010
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Contents |
Andreas
Transfer of ProtA⋅His to pEX
Digestion
| Sample | |
|---|---|
| 10X FastDigest buffer | 1 |
| Plasmid DNA | 7 |
| dH2O | 0 |
| FD XbaI | 1 |
| FD PstI | 1 |
| 10 μl |
- Incubation: 37 °C, 0:30
- Inactivation: 80 °C, 3:00
De-phosphorylated pre-digested/extracted pEX vector:
- 8 μl extracted and digested (X+P) pEX vector DNA
- 1 μl FD buffer
- 1 μl FastAP (alkaline phosphatase)
- Incubation: 37 °C, 0:30
- Inactivation: 80 °C, 3:00
Ligation
| 10X T4 Ligase buffer | 2 |
| Vector DNA | 1 |
| Insert DNA | 11 |
| dH2O | 5 |
| T4 DNA ligase | 1 |
| 20 μl |
|---|
- Incubation: 22 °C, 1 h
Transformation
Including three more transformations
Modified quick-transformation protocol:
- 30 min on ice
- 50 μl BL21
- 2 μl pEX.ProtA⋅his ligation mix
- 0.5 μl pEX.IgGp
- 0.5 μl pEX.SOD.RyC
- 0.5 μl pEX.hS.RyC
PCR verification for Uppsala-Sweden team
Helping the Uppsala team with a PCR verification of one of their assemblies.
Summed up their total construct length in pSB1x3 to 6566 bp.
- K1: C2 & C4 (x2)
- K2: C2 & C5 (x2)
Standard colony PCR settings:
- Elongation time: 10 min
- Annealing temp: 55 °C and 60 °C
PCR run ON.
