Paris/July 30
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== Ligations == | == Ligations == | ||
Merci Eimad | Merci Eimad | ||
| + | |||
| + | ==MiniPreps== | ||
| + | * L1''.1, L1''.2 | ||
| + | * L6''.1, L6''.2 | ||
Revision as of 10:15, 31 July 2007
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
Merci Eimad
MiniPreps
- L1.1, L1.2
- L6.1, L6.2
