Paris/July 30
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Contents |
Sequences
All the biobricks that we got from the plate and all the ligations that succeeded up to now.
Colonies PCR screen
On colony PCRs performed to screen for correct ligation reactions (performed on 27/07/07):
10 clones of L1 ligation reactions, 6 clones of L6 ligation reactions
preparation of the following PCR mix
- 18x (12.5µL 2xreaction mix)= 225
- 18x (0.5µL oligo O18 VF2 10µM)=9
- 18x (0.5µL oligo O19 VR 10µM)=9
- 12µL H20+colony (10min 100°)
PCRs
| PCR : P5 lox66-DapAColi | ||||||
|---|---|---|---|---|---|---|
| PCR Settings | Buffer (5x) | 5x 10µL | Expected size | |||
| Annealing (°C) | MgCl2 10µM | 10µM 0µL | 985 | |||
| 55 | dNTP 10µM | 10µM 1µL | Success | |||
| Time Elongation | Oligo F 10µM | O6 | 2.5µL | |||
| 2m00' | Oligo R 10µM | O7 | 2.5µL | Image (click to enlarge) | ||
| Number cycles | Water | 34µl | [[Image:|30px]] | |||
| 30 | Polymerase | Phusion 0.5µL | Band (0=ladder) | |||
| DNA | Toothpick in MG16655 Glycerol | |||||
| PCR : P6 Lox66-DapASubtilis | ||||||
|---|---|---|---|---|---|---|
| PCR Settings | Buffer (5x) | 5x 10µL | Expected size | |||
| Annealing (°C) | MgCl2 10µM | 10µM 0µL | 979 | |||
| 55 | dNTP 10µM | 10µM 1µL | Success | |||
| Time Elongation | Oligo F 10µM | O8 | 2.5µL | |||
| 2m00' | Oligo R 10µM | O9 | 2.5µL | Image (click to enlarge) | ||
| Number cycles | Water | 34µl | [[Image:|30px]] | |||
| 30 | Polymerase | Phusion 0.5µL | Band (0=ladder) | |||
| DNA | 0.5µl Subtilis DNA | |||||
| PCR : P7 Lox71-FtsZ | ||||||
|---|---|---|---|---|---|---|
| PCR Settings | Buffer (5x) | 5x 10µL | Expected size | |||
| Annealing (°C) | MgCl2 10µM | 10µM 0µL | 1260 | |||
| 55 | dNTP 10µM | 10µM 1µL | Success | |||
| Time Elongation | Oligo F 10µM | O1 | 2.5µL | |||
| 2m00' | Oligo R 10µM | O2 | 2.5µL | Image (click to enlarge) | ||
| Number cycles | Water | 34µl | [[Image:|30px]] | |||
| 30 | Polymerase | Phusion 0.5µL | Band (0=ladder) | |||
| DNA | Toothpick in MG16655 Glycerol | |||||
| PCR : P8 RBS-DapAColi | ||||||
|---|---|---|---|---|---|---|
| PCR Settings | Buffer (5x) | 5x 10µL | Expected size | |||
| Annealing (°C) | MgCl2 10µM | 10µM 0µL | 952 | |||
| 55 | dNTP 10µM | 10µM 1µL | Success | |||
| Time Elongation | Oligo F 10µM | O20 | 2.5µL | |||
| 2m00' | Oligo R 10µM | O7 | 2.5µL | Image (click to enlarge) | ||
| Number cycles | Water | 34µl | [[Image:|30px]] | |||
| 30 | Polymerase | Phusion 0.5µL | Band (0=ladder) | |||
| DNA | Toothpick in MG16655 Glycerol | |||||
| PCR : P11 Lox71-KmR-Lox66 | ||||||
|---|---|---|---|---|---|---|
| PCR Settings | Buffer (5x) | 5x 10µL | Expected size | |||
| Annealing (°C) | MgCl2 10µM | 10µM 0µL | 1084 | |||
| 55 | dNTP 10µM | 10µM 1µL | Success | |||
| Time Elongation | Oligo F 10µM | O10 | 2.5µL | |||
| 2m00' | Oligo R 10µM | O11 | 2.5µL | Image (click to enlarge) | ||
| Number cycles | Water | 34µl | [[Image:|30px]] | |||
| 30 | Polymerase | Phusion 0.5µL | Band (0=ladder) | |||
| DNA | 0.5 µl MP12 | |||||
Digestion reactions
The following digestion reactions have been performed:
| Digestion Products | ||||||||
|---|---|---|---|---|---|---|---|---|
| Number | Product Name | Matrix Name | Enzyme 1 | Enzyme 2 | size | Description | ||
| D19' | BB dig. lox66DapAsubtilis | lox66-DapAsubtilis P6' PCR product | EcoRI | PstI | ~960 | PCR product digested for cloning as a BioBrick | ||
| D21' | bw-insert lox66-DapAsubtilis | lox66-DapAsubtilis P6' PCR product | XbaI | PstI | ~960 | PCR product digested for cloning as a BI | ||
| D27' | lox71-ftsZ bw-insert | lox71-ftsZ PCR product P7 | XbaI | PstI | lox71-ftsZ(unmutated) ready for use as a backward insert | |||
| D33 | RBS(B0030)CreORF | L6' (clone 1 & 2) | XbaI | PstI | RBS-Cre digestion for use as a BI | |||
| D34 | lox66 | L9' lox66 | SpeI | PstI | Biobrick plasmid carrying lox66 digested as a BV | |||
| D35 | lox71 | L10' lox71 | SpeI | PstI | Biobrick plasmid carrying lox71 digested as a BV | |||
Ligations
| Ligations | ||||||
|---|---|---|---|---|---|---|
| Number | Insert | Insert Volume (µL) | Vector | Vector Volume (µL) | Comments | Number of colonies |
| Control 1 | MP3.1 digested with EcoRI (D0) | 2 | control of ligation efficiency | |||
| a faire | MP3.1 digested with EcoRI (D0) | 2 | without ligase in the mix: control of digestion efficiency | |||
| Control 2 | D35 | 2µl | control of D35 digestion | 1 | ||
| Control 3 | D34 | 2µl | control of D34 digestion | 2 | ||
| L3 | D19 (lox66dapAsubtilis) | 4 | D22.1 (pSB1A2 dig. EcoRI&PstI) | 2 | Insertion of lox66DapASubtilis as a biobrick | |
| L5 | D21’(lox66DapASubtilis) | 4 | D1 (j23100 digested for CDS insertion) | 2 | construction of the biobrick J23100-lox66dapAsubtilis | 0 |
| L7 | D27' (lox-ftsZ) | 4 | D1 (j23100 digested for CDS insertion) | 2 | construction of the biobrick J23100- lox-ftsZ | 0 |
| L8 | D27' (lox-ftsZ) | 4 | D3 (j23107 digested for CDS insertion) | 2 | construction of the biobrick J23107- lox-ftsZ | 0 |
| L9 | lox66 [O14+O15] (2µM) | 2 | D22.2 (pSB1A2 Eco, Pst) | 2 | lox66 cloned as a biobrick in pSB1A2 | |
| L10 | lox71 [O16+O17] (2µM) | 2 | D22.2 (pSB1A2 dig. EcoRI & PstI) | 2 | lox71 cloned as a biobrick in pSB1A2 | 0 |
| L11 | D21’(lox66dapAsubtilis BI) | 4 | D3 (j23107 digested for CDS insertion) | 2 | construction of the biobrick: J23107 promotor-lox66dapAsubtilis | 1 |
| L12 | D26.1 (gfp-tripart BI) | 4 | D35 (lox66 plasmid digested as a BV) | 2 | construction of the biobrick lox66-gfp | |
| L13 | D13.2 (mRFP de pJ23100) | 4 | D34 (lox71 plasmid digested as a BV) | 2 | lox71 cloned as a biobrick | 0 |
| L14 | D33.1 (RBS-Cre BI) | 4 | D1 (j23100 digested for CDS insertion) | 2 | J23100-RBS-Cre | |
| L15 | D33.1 (RBS-Cre BI) | 4 | D3 (j23107 digested for CDS insertion) | 2 | J23107-RBS-Cre | 0 |
| L16 | D32 (lox71-ftsAftsZ BI) | 4 | D1 (j23100 digested for CDS insertion) | 2 | J23100-lox71-ftsAftsZ | 0 |
| L17 | D32 (lox71-ftsAftsZ BI) | 4 | D3 (j23107 digested for CDS insertion) | 2 | J23107-lox71-ftsAftsZ | 0 |
| L18 | D31 (RBSdapAsubtilis BI) | 4 | D1 (j23100 digested for CDS insertion) | 2 | J23100-RBSdapAsubtilis | 0 |
| L19 | D31 (RBSdapAsubtilis BI) | 4 | D3 (j23107 digested for CDS insertion) | 2 | J23107-RBSdapAsubtilis | 0 |
| L20 | D30 (RBSdapAsubtilis dig. BB) | 4 | D22.2 (pSB1A2 Eco, Pst) | 2 | 0 | |
| L21 | D29 | 4 | D1 (j23100 digested for CDS insertion) | 2 | 0 | |
| L22 | D29 | 4 | D3 (j23107 digested for CDS insertion) | 2 | 0 | |
| L23 | D28 (RBSdapAcoli dig.BB) | 4 | D22.2 (pSB1A2 Eco, Pst) | 2 | 1 | |
| L24 | D21’(lox66dapAsubtilis BI) | 4 | D3 (j23107 digested for CDS insertion) | 2 | 0 | |
| L25 | D36 (lox71-KmR-lox66 dig. BB) | 4 | D22.2 (pSB1A2 dig. EcoRI & PstI) | 2 | 1 | |
Ligation mix:
- 1µl 10x buffer
- 0.5µl Ligase
- 2µL plasmid DNA solution
- 4µL insert DNA solution
Reaction in 10µl
ON @ 4°C
MiniPreps
- L1".1, L1".2
- L6".1, L6".2
