Paris/July 30
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Revision as of 18:52, 30 July 2007 by David.bikard (Talk | contribs)
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 | |||
