Team:Stockholm/25 September 2010
From 2010.igem.org
Contents |
Andreas
Cloning and assembly
Digestion
Further digestions in addition to the ones performed 24/9.
pK.N-TAT⋅ SH (4) X+P | pK.N-LMWP⋅ SH (1) X+P | pK.N-LMWP⋅ SH (1) S+P | |
---|---|---|---|
10X FastDigest buffer | 2 | 2 | 2 |
DNA (1 μg) | 8 | 4 | 4 |
dH2O | 8 | 12 | 12 |
FD XbaI | 1 | 1 | 0 |
FD SpeI | 0 | 0 | 1 |
FD PstI | 1 | 1 | 1 |
20 μl | 20 μl | 20 μl |
- Incubation: 37 °C, 1 h
- Inactivation: 80 °C, 30 min (including digestion samples from 24/9)
Ligation
Underlined sequences relevant for assembly
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
---|---|---|---|---|---|---|---|---|
10X T4 Ligase buffer | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 |
Vector DNA (50 ng) | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
Insert DNA | 5 | 5 | 5 | 5 | 4 | 4 | 4 | 4 |
dH2O | 11 | 11 | 11 | 11 | 12 | 12 | 12 | 12 |
T4 DNA ligase | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
20 μl | 20 μl | 20 μl | 20 μl | 20 μl | 20 μl | 20 μl | 20 μl |
- pEX.N-LMWP⋅SOD⋅His (K)
- pSB1K3.N-LMWP⋅SOD⋅His X+P
- pEX.RFP X+P
- pEX.N-TAT⋅SOD⋅His
- pSB1K3.N-TAT⋅SOD⋅His X+P
- pEX.RFP X+P
- pEX.N-Tra10⋅SOD⋅His
- pSB1K3.N-Tra10⋅SOD⋅His X+P
- pEX.RFP X+P
- pEX.N-LMWP⋅SOD⋅His (C)
- pSB1C3.N-LMWP⋅SOD⋅His X+P
- pEX.RFP X+P
- pSB1K3.N-LMWP⋅SOD⋅His.RBS.yCCS
- pSB1K3.N-LMWP⋅SOD⋅His S+P
- pSB1A2.RBS.yCCS X+P
- pSB1K3.N-TAT⋅SOD⋅His.RBS.yCCS
- pSB1K3.N-TAT⋅SOD⋅His S+P
- pSB1A2.RBS.yCCS X+P
- pSB1K3.N-Tra10⋅SOD⋅His.RBS.yCCS
- pSB1K3.N-Tra10⋅SOD⋅His S+P
- pSB1A2.RBS.yCCS X+P
- pSB1C3.N-LMWP⋅SOD⋅His.RBS.yCCS
- pSB1C3.N-LMWP⋅SOD⋅His S+P
- pSB1A2.RBS.yCCS X+P
Transformation
- Standard transformation according to protocol.
- 1 μl ligation mix
- Amp 100, Cm 25 or Km 50 plates
- 50 μl IPTG (pEX constructs, Amp 100 plates)
Sequencing preparation
Prepared samples for sequencing to be sent on Monday.
Construct | Primer | Sample name | Sequencing code (edited 27/9) |
---|---|---|---|
pEX.N-TAT⋅SOD⋅His 3 | pEXf | pEX.nTAT*SH3_pEXf | ASB0045 614 |
pEX.N-TAT⋅SOD⋅His 4 | pEXf | pEX.nTAT*SH4_pEXf | ASB0045 615 |
pSB1A2.RBS.yCCS 3 | VF2 | pA.RBS.yCCS3_VF2 | ASB0045 603 |
pSB1A2.RBS.yCCS 4 | VF2 | pA.RBS.yCCS4_VF2 | ASB0045 604 |
pSB1K3.N-TAT⋅SOD⋅His 4 | VF2 | pK.nTAT*SH4_VF2 | ASB0045 608 |
pSB1K3.N-TAT⋅SOD⋅His 5 | VF2 | pK.nTAT*SH5_VF2 | ASB0045 609 |
pSB1K3.N-Tra10⋅SOD⋅His 5 | VF2 | pK.nTra10*SH5_VF2 | ASB0045 610 |
pSB1K3.N-LMWP⋅SOD⋅His 1 | VF2 | pK.nLMWP*SH1_VF2 | ASB0045 607 |
pSB1C3.N-LMWP⋅SOD⋅His 1 | VF2 | pC.nLMWP*SH1_VF2 | ASB0045 605 |
pSB1C3.N-LMWP⋅SOD⋅His 4 | VF2 | pC.nLMWP*SH4_VF2 | ASB0045 606 |
pEX.SOD | pEXf | pEX.SOD_pEXf | ASB0045 611 |
pEX.yCCS 5 | pEXf | pEX.yCCS5_pEXf | ASB0045 616 |
pEX.SOD⋅His | pEXf | pEX.SH_pEXf | ASB0045 612 |
pEX.His⋅SOD | pEXf | pEX.HS_pEXf | ASB0045 613 |
BL21 transformation
Transformed BL21 cells with not-yet-verified pEX.N-TAT⋅SOD⋅His plasmid samples 3 & 4
- Standard transformation according to protocol.
- 1 μl ligation mix
- 100 μl cell aliquot divided in 2x50 μl to lower number of colonies after transformation.
- Amp 100 plates
- 1 μl ligation mix
Preparation of LB agar plates
Made new Cm (25 μg/ml) and Km (50 μg/ml) LB agar plates.
- 10X Cm 25
- 10X Km 50
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