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Team:Stockholm/25 September 2010
From 2010.igem.org
(Difference between revisions)
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!Primer | !Primer | ||
!Sample name | !Sample name | ||
| + | !Sequencing code<br />(edited 27/9) | ||
|- | |- | ||
|pEX.N-TAT⋅SOD⋅His 3 | |pEX.N-TAT⋅SOD⋅His 3 | ||
|pEXf | |pEXf | ||
|pEX.nTAT*SH3_pEXf | |pEX.nTAT*SH3_pEXf | ||
| + | |ASB0045 614 | ||
|- | |- | ||
|pEX.N-TAT⋅SOD⋅His 4 | |pEX.N-TAT⋅SOD⋅His 4 | ||
|pEXf | |pEXf | ||
|pEX.nTAT*SH4_pEXf | |pEX.nTAT*SH4_pEXf | ||
| + | |ASB0045 615 | ||
|- | |- | ||
|pSB1A2.RBS.yCCS 3 | |pSB1A2.RBS.yCCS 3 | ||
|VF2 | |VF2 | ||
|pA.RBS.yCCS3_VF2 | |pA.RBS.yCCS3_VF2 | ||
| + | |ASB0045 603 | ||
|- | |- | ||
|pSB1A2.RBS.yCCS 4 | |pSB1A2.RBS.yCCS 4 | ||
|VF2 | |VF2 | ||
|pA.RBS.yCCS4_VF2 | |pA.RBS.yCCS4_VF2 | ||
| + | |ASB0045 604 | ||
|- | |- | ||
|pSB1K3.N-TAT⋅SOD⋅His 4 | |pSB1K3.N-TAT⋅SOD⋅His 4 | ||
|VF2 | |VF2 | ||
|pK.nTAT*SH4_VF2 | |pK.nTAT*SH4_VF2 | ||
| + | |ASB0045 608 | ||
|- | |- | ||
|pSB1K3.N-TAT⋅SOD⋅His 5 | |pSB1K3.N-TAT⋅SOD⋅His 5 | ||
|VF2 | |VF2 | ||
|pK.nTAT*SH5_VF2 | |pK.nTAT*SH5_VF2 | ||
| + | |ASB0045 609 | ||
|- | |- | ||
|pSB1K3.N-Tra10⋅SOD⋅His 5 | |pSB1K3.N-Tra10⋅SOD⋅His 5 | ||
|VF2 | |VF2 | ||
|pK.nTra10*SH5_VF2 | |pK.nTra10*SH5_VF2 | ||
| + | |ASB0045 610 | ||
|- | |- | ||
|pSB1K3.N-LMWP⋅SOD⋅His 1 | |pSB1K3.N-LMWP⋅SOD⋅His 1 | ||
|VF2 | |VF2 | ||
|pK.nLMWP*SH1_VF2 | |pK.nLMWP*SH1_VF2 | ||
| + | |ASB0045 607 | ||
|- | |- | ||
|pSB1C3.N-LMWP⋅SOD⋅His 1 | |pSB1C3.N-LMWP⋅SOD⋅His 1 | ||
|VF2 | |VF2 | ||
|pC.nLMWP*SH1_VF2 | |pC.nLMWP*SH1_VF2 | ||
| + | |ASB0045 605 | ||
|- | |- | ||
|pSB1C3.N-LMWP⋅SOD⋅His 4 | |pSB1C3.N-LMWP⋅SOD⋅His 4 | ||
|VF2 | |VF2 | ||
|pC.nLMWP*SH4_VF2 | |pC.nLMWP*SH4_VF2 | ||
| + | |ASB0045 606 | ||
|- | |- | ||
|pEX.SOD | |pEX.SOD | ||
|pEXf | |pEXf | ||
|pEX.SOD_pEXf | |pEX.SOD_pEXf | ||
| + | |ASB0045 611 | ||
|- | |- | ||
|pEX.yCCS 5 | |pEX.yCCS 5 | ||
|pEXf | |pEXf | ||
|pEX.yCCS5_pEXf | |pEX.yCCS5_pEXf | ||
| + | |ASB0045 616 | ||
|- | |- | ||
|pEX.SOD⋅His | |pEX.SOD⋅His | ||
|pEXf | |pEXf | ||
|pEX.SH_pEXf | |pEX.SH_pEXf | ||
| + | |ASB0045 612 | ||
|- | |- | ||
|pEX.His⋅SOD | |pEX.His⋅SOD | ||
|pEXf | |pEXf | ||
|pEX.HS_pEXf | |pEX.HS_pEXf | ||
| + | |ASB0045 613 | ||
|} | |} | ||
Revision as of 17:23, 27 September 2010
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 (4) X+P | pK.N-LMWP⋅ SH (4) 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.SOD X+P
- pEX.N-TAT⋅SOD⋅His
- pSB1K3.N-TAT⋅SOD⋅His X+P
- pEX.SOD X+P
- pEX.N-Tra10⋅SOD⋅His
- pSB1K3.N-Tra10⋅SOD⋅His X+P
- pEX.SOD X+P
- pEX.N-LMWP⋅SOD⋅His (C)
- pSB1C3.N-LMWP⋅SOD⋅His X+P
- pEX.SOD 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
