Team:Stockholm/18 October 2010
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==Andreas== | ==Andreas== | ||
+ | |||
+ | ===Transfer of ProtA⋅His to pEX=== | ||
+ | |||
+ | ====Digestion==== | ||
+ | |||
+ | {|border="1" cellpadding="1" cellspacing="0" | ||
+ | | | ||
+ | !width="50"|Sample | ||
+ | |- | ||
+ | |10X FastDigest buffer | ||
+ | |align="center"|1 | ||
+ | |- | ||
+ | |Plasmid DNA | ||
+ | |align="center"|7 | ||
+ | |- | ||
+ | |dH<sub>2</sub>O | ||
+ | |align="center"|0 | ||
+ | |- | ||
+ | |FD XbaI | ||
+ | |align="center"|1 | ||
+ | |- | ||
+ | |FD PstI | ||
+ | |align="center"|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==== | ||
+ | {|border="1" cellpadding="1" cellspacing="0" | ||
+ | |10X T4 Ligase buffer | ||
+ | |align="center"|2 | ||
+ | |- | ||
+ | |Vector DNA | ||
+ | |align="center"|1 | ||
+ | |- | ||
+ | |Insert DNA | ||
+ | |align="center"|11 | ||
+ | |- | ||
+ | |dH<sub>2</sub>O | ||
+ | |align="center"|5 | ||
+ | |- | ||
+ | |T4 DNA ligase | ||
+ | |align="center"|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 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. |
Revision as of 18:20, 18 October 2010
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 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.