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Template:Kyoto/Notebook/Aug 19
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(Created page with "==Aug 19== ===Colony PCR=== <div class="experiment"> <span class="author">Kojima</span> {| class="wikitable" !|Sample||base pair |- |8/18 J23100 control -(1)||1142 |- |8/18 J2310...")
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Revision as of 22:38, 16 September 2013
Contents |
Aug 19
Colony PCR
| Sample | base pair |
|---|---|
| 8/18 J23100 control -(1) | 1142 |
| 8/18 J23100-RBS-GFP-DT -(1) | 1156 |
| 8/18 J23100-RBS-GFP-DT -(2) | 1156 |
| 8/18 J23100-RBS-luxR-DT -(1) | 1271 |
| 8/18 J23100-RBS-luxR-DT -(2) | 1271 |
| 8/18 J23100-RBS-lacZα-DT -(1) | 670 |
| 8/18 J23100-RBS-lacZα-DT -(2) | 670 |
| 8/18 RBS-lysis3 -(1) | 997 |
| 8/18 RBS-lysis3 -(2) | 997 |
| 8/18 P0440(RBS-tetR-DT) -(1) | 1154 |
| 8/18 P0440(RBS-tetR-DT) -(2) | 1154 |
| negative control | -- |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 1min | 30cycles |
File:Igku Aug19electrophoresis1.png
| Sample | base pair |
|---|---|
| 8/18 RBS control -(1) | -- |
| 8/18 R0040(Ptet) -(1) | 686 |
| 8/18 R0040(Ptet) -(2) | 686 |
| negative control | -- |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 30s | 30cycles |
Gel Extraction
| Lane | DNA | Enzyme |
|---|---|---|
| 1 | 100bp ladder | -- |
| 3 | 8/18 K117000(lysis2) -1 | XbaI & PstI |
| 5 | 8/18 K117000(lysis2) -1 | XbaI & PstI |
File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4
| Name | concentration[µg/mL] | 260/280 | 260/230 |
|---|---|---|---|
| 8/18 lysis2-1(XbaI & PstI) | 3.1 | 3.64 | 0.19 |
| 8/18 lysis2-1(XbaI & PstI) | 3.4 | 1.89 | 0.13 |
LB Medium Plate
| volume | 200ml |
|---|---|
| Bacto T2ypton | 2g |
| Bacto yeast extract | 1g |
| NaCl | 1g |
| Agar Pouder | 2gă |
Restriction Enzyme Digestion
| lysis2-1 | XbaI | PstI | BSA | 10xbuffer | MilliQ | total |
|---|---|---|---|---|---|---|
| 10µL | 1µL | 1µL | 3µL | 3µL | 12µL | 30µL |
| 2µL | 0.2µL | -- | 1µL | 1µL | 5.8µL | 10µL |
| 2µL | -- | 0.2µL | 1µL | 1µL | 5.8µL | 10µL |
| 2µLL | -- | -- | 1µL | 1µ | 6µL | 10µL |
- at 37°C, for 1h
| 8/19 lysis1-1 | 10xbuffer | XbaI | PstI | BSA | MilliQ | total |
|---|---|---|---|---|---|---|
| 2µL | 2µL | 0.5µL | 0.8µL | 2µL | 13µL | 20µL |
| 0.5µL | 1µL | 0.2µL | -- | 1µL | 7.3µL | 10µL |
| 0.5µL | 1µL | -- | 0.2µL | 1µL | 7.3µL | 10µL |
| 0.5µL | 1µL | -- | -- | 1µL | 7.5µL | 10µL |
- at 37°C, for 1h
Ligation
| Name | NC | |
|---|---|---|
| RBS | 0.3 | 0.3 |
| lysis2 | 7.4 | -- |
| MilliQ | -- | 7.4 |
Electrophoresis
| Lane | Sample | Enzyme1 | Enzyme2 |
|---|---|---|---|
| 1 | 100bp ladder | -- | -- |
| 2 | 8/19 lysis1 | XbaI | PstI |
| 3 | 8/19 lysis1 | XbaI | -- |
| 4 | 8/19 lysis1 | -- | PstI |
| 5 | 8/19 lysis1 | -- | -- |
| 6 | 8/19 lysis1(Gel Extraction Product) | XbaI | PstI |
Transformation
| Name | Well | Sample | Competent Cells | Plate |
|---|---|---|---|---|
| 8/18 Plux+RBS-GFP-DT | -- | 2µL | 20µL | CP |
| 8/18 Plux(NC) | -- | 2µL | 20µL | CP |
| 8/19 RBS+lysis2 | -- | 2µL | 20µL | Amp |
| 8/15 Pbad-araC | Plate5-14N | 2µL | 20µL | Kan |
- Incubate at 37°C
Gel Extraction
| Lane | DNA | Enzyme |
|---|---|---|
| 1 | 100bp ladder | -- |
| 2 | 8/19 lysis1 ① | XbaI & PstI |
[fig before] [fig after]
| Name | concentration[µg/mL] | 260/280 | 260/230 |
|---|---|---|---|
| 8/19 lysis1 ①(XbaI & PstI) | 87.8 | 1.06 | 0.82 |
Liquid Culture
| Sample | medium |
|---|---|
| 8/18 Ptet -1 | Plusgrow medium(+CP) |
| 8/18 Ptet -2 | Plusgrow medium(+CP) |
| 8/18 RBS-tetR-DT -1 | Plusgrow medium(+CP)ă |
| 8/18 RBS-tetR-DT -2 | Plusgrow medium(+CP)ă |
| 8/18 J23100-GFP -1 | Plusgrow medium(+Amp) |
| 8/18 J23100-luxR -1 | Plusgrow medium(+Amp) |
| 8/18 J23100-luxR -2 | Plusgrow medium(+Amp) |
| 8/18 J23100-lacZα -1 | Plusgrow medium(+Amp) |
| 8/18 RBS-lysis3 -1 | Plusgrow medium(+Amp) |
| 8/18 RBS-lysis3 -2 | Plusgrow medium(+Amp) |
Master Plate
| Number | Use LB plate(+Amp) |
|---|---|
| 1 | 8/18 J23100-GFP -1 |
| 2 | 8/18 J23100-luxR -1 |
| 3 | 8/18 J23100-luxR -2 |
| 4 | 8/18 J23100-lacZα -1 |
| 5 | 8/18 RBS-lysis3 -1 |
| 6 | 8/18 RBS-lysis3 -2 |
| Number | Use LB plate(+CP) |
|---|---|
| 1 | 8/18 Ptet -1 |
| 1 | 8/18 Ptet -2 |
| 3 | 8/18 RBS-tetR-DT -1 |
| 4 | 8/18 RBS-tetR-DT -2 |
