Team:Stockholm/30 September 2010
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==Andreas== | ==Andreas== | ||
+ | |||
+ | ===Transfer of nCPP⋅SOD⋅His.RBS.yCCS operon to pEX=== | ||
+ | |||
+ | ====Digestions==== | ||
+ | #pSB1K3.nTAT⋅SOD⋅His.RBS.yCCS | ||
+ | #*Clones 2 & 3 | ||
+ | #pSB1K3.nTra10⋅SOD⋅His.RBS.yCCS | ||
+ | #*Clones 1 & 2 | ||
+ | #pSB1K3.nLMWP⋅SOD⋅His.RBS.yCCS | ||
+ | #*Clones 2 & 3 | ||
+ | |||
+ | |||
+ | {|border="1" cellpadding="1" cellspacing="0" | ||
+ | | | ||
+ | |align="center" width="50"|'''1''':2 | ||
+ | |align="center" width="50"|'''1''':3 | ||
+ | |align="center" width="50"|'''2''':1 | ||
+ | |align="center" width="50"|'''2''':2 | ||
+ | |align="center" width="50"|'''3''':2 | ||
+ | |align="center" width="50"|'''3''':3 | ||
+ | |- | ||
+ | |10X FastDigest buffer | ||
+ | |align="center"|2 | ||
+ | |align="center"|2 | ||
+ | |align="center"|2 | ||
+ | |align="center"|2 | ||
+ | |align="center"|2 | ||
+ | |align="center"|2 | ||
+ | |- | ||
+ | |DNA (1 μg) | ||
+ | |align="center"|5.2 | ||
+ | |align="center"|4.1 | ||
+ | |align="center"|2.5 | ||
+ | |align="center"|3.3 | ||
+ | |align="center"|4 | ||
+ | |align="center"|4.1 | ||
+ | |- | ||
+ | |dH<sub>2</sub>O | ||
+ | |align="center"|10.8 | ||
+ | |align="center"|11.9 | ||
+ | |align="center"|13.5 | ||
+ | |align="center"|12.7 | ||
+ | |align="center"|12 | ||
+ | |align="center"|11.9 | ||
+ | |- | ||
+ | |FD XbaI | ||
+ | |align="center"|1 | ||
+ | |align="center"|1 | ||
+ | |align="center"|1 | ||
+ | |align="center"|1 | ||
+ | |align="center"|1 | ||
+ | |align="center"|1 | ||
+ | |- | ||
+ | |FD PstI | ||
+ | |align="center"|1 | ||
+ | |align="center"|1 | ||
+ | |align="center"|1 | ||
+ | |align="center"|1 | ||
+ | |align="center"|1 | ||
+ | |align="center"|1 | ||
+ | |- | ||
+ | | | ||
+ | !20 μl | ||
+ | !20 μl | ||
+ | !20 μl | ||
+ | !20 μl | ||
+ | !20 μl | ||
+ | !20 μl | ||
+ | |} | ||
+ | *Incubation: 37 °C, 1.45 | ||
+ | *Inactivation: 80 °C, 20 min | ||
+ | |||
+ | ====Ligations==== | ||
+ | *Vector: [Dig pEX.RFP X+P | ||
+ | |||
+ | {|border="1" cellpadding="1" cellspacing="0" | ||
+ | | | ||
+ | |align="center" width="50"|pEX.'''1''':2 | ||
+ | |align="center" width="50"|pEX.'''1''':3 | ||
+ | |align="center" width="50"|pEX.'''2''':1 | ||
+ | |align="center" width="50"|pEX.'''2''':2 | ||
+ | |align="center" width="50"|pEX.'''3''':2 | ||
+ | |align="center" width="50"|pEX.'''3''':3 | ||
+ | |- | ||
+ | |10X T4 Ligase buffer | ||
+ | |align="center"|2 | ||
+ | |align="center"|2 | ||
+ | |align="center"|2 | ||
+ | |align="center"|2 | ||
+ | |align="center"|2 | ||
+ | |align="center"|2 | ||
+ | |- | ||
+ | |Vector DNA | ||
+ | |align="center"|1.5 | ||
+ | |align="center"|1.5 | ||
+ | |align="center"|1.5 | ||
+ | |align="center"|1.5 | ||
+ | |align="center"|1.5 | ||
+ | |align="center"|1.5 | ||
+ | |- | ||
+ | |Insert DNA | ||
+ | |align="center"|8 | ||
+ | |align="center"|8 | ||
+ | |align="center"|8 | ||
+ | |align="center"|8 | ||
+ | |align="center"|8 | ||
+ | |align="center"|8 | ||
+ | |- | ||
+ | |dH<sub>2</sub>O | ||
+ | |align="center"|7.5 | ||
+ | |align="center"|7.5 | ||
+ | |align="center"|7.5 | ||
+ | |align="center"|7.5 | ||
+ | |align="center"|7.5 | ||
+ | |align="center"|7.5 | ||
+ | |- | ||
+ | |T4 DNA ligase | ||
+ | |align="center"|1 | ||
+ | |align="center"|1 | ||
+ | |align="center"|1 | ||
+ | |align="center"|1 | ||
+ | |align="center"|1 | ||
+ | |align="center"|1 | ||
+ | |- | ||
+ | | | ||
+ | !20 μl | ||
+ | !20 μl | ||
+ | !20 μl | ||
+ | !20 μl | ||
+ | !20 μl | ||
+ | !20 μl | ||
+ | |} | ||
+ | *Incubation: 22 °C, 15 min | ||
+ | |||
+ | ====Transformation==== | ||
+ | Quick transformation | ||
+ | *1 μl ligation mix | ||
+ | *50 μl 0.1 M IPTG | ||
+ | **pEX.1:2 (pEX.nTAT⋅SOD⋅His.RBS.yCCS 2) | ||
+ | **pEX.1:3 (pEX.nTAT⋅SOD⋅His.RBS.yCCS 2) | ||
+ | **pEX.2:1 (pEX.nTra10⋅SOD⋅His.RBS.yCCS 1) | ||
+ | **pEX.2:2 (pEX.nTra10⋅SOD⋅His.RBS.yCCS 2) | ||
+ | **pEX.3:2 (pEX.nLMWP⋅SOD⋅His.RBS.yCCS 2) | ||
+ | **pEX.3:3 (pEX.nLMWP⋅SOD⋅His.RBS.yCCS 3) | ||
+ | |||
+ | ===Transformation of BL21=== | ||
+ | Quick transformation | ||
+ | *50 μl competent cells | ||
+ | *0.5 μl plasmid | ||
+ | **pEX.nTra10⋅SOD⋅His | ||
+ | **pEX.nLMWP⋅SOD⋅His |
Revision as of 19:34, 30 September 2010
Contents |
Andreas
Transfer of nCPP⋅SOD⋅His.RBS.yCCS operon to pEX
Digestions
- pSB1K3.nTAT⋅SOD⋅His.RBS.yCCS
- Clones 2 & 3
- pSB1K3.nTra10⋅SOD⋅His.RBS.yCCS
- Clones 1 & 2
- pSB1K3.nLMWP⋅SOD⋅His.RBS.yCCS
- Clones 2 & 3
1:2 | 1:3 | 2:1 | 2:2 | 3:2 | 3:3 | |
10X FastDigest buffer | 2 | 2 | 2 | 2 | 2 | 2 |
DNA (1 μg) | 5.2 | 4.1 | 2.5 | 3.3 | 4 | 4.1 |
dH2O | 10.8 | 11.9 | 13.5 | 12.7 | 12 | 11.9 |
FD XbaI | 1 | 1 | 1 | 1 | 1 | 1 |
FD PstI | 1 | 1 | 1 | 1 | 1 | 1 |
20 μl | 20 μl | 20 μl | 20 μl | 20 μl | 20 μl |
---|
- Incubation: 37 °C, 1.45
- Inactivation: 80 °C, 20 min
Ligations
- Vector: [Dig pEX.RFP X+P
pEX.1:2 | pEX.1:3 | pEX.2:1 | pEX.2:2 | pEX.3:2 | pEX.3:3 | |
10X T4 Ligase buffer | 2 | 2 | 2 | 2 | 2 | 2 |
Vector DNA | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 |
Insert DNA | 8 | 8 | 8 | 8 | 8 | 8 |
dH2O | 7.5 | 7.5 | 7.5 | 7.5 | 7.5 | 7.5 |
T4 DNA ligase | 1 | 1 | 1 | 1 | 1 | 1 |
20 μl | 20 μl | 20 μl | 20 μl | 20 μl | 20 μl |
---|
- Incubation: 22 °C, 15 min
Transformation
Quick transformation
- 1 μl ligation mix
- 50 μl 0.1 M IPTG
- pEX.1:2 (pEX.nTAT⋅SOD⋅His.RBS.yCCS 2)
- pEX.1:3 (pEX.nTAT⋅SOD⋅His.RBS.yCCS 2)
- pEX.2:1 (pEX.nTra10⋅SOD⋅His.RBS.yCCS 1)
- pEX.2:2 (pEX.nTra10⋅SOD⋅His.RBS.yCCS 2)
- pEX.3:2 (pEX.nLMWP⋅SOD⋅His.RBS.yCCS 2)
- pEX.3:3 (pEX.nLMWP⋅SOD⋅His.RBS.yCCS 3)
Transformation of BL21
Quick transformation
- 50 μl competent cells
- 0.5 μl plasmid
- pEX.nTra10⋅SOD⋅His
- pEX.nLMWP⋅SOD⋅His