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Template:Kyoto/Notebook/Sep 9
From 2013.igem.org
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<span class="author">Tatsui and Yoshida</span> | <span class="author">Tatsui and Yoshida</span> | ||
{| class="wikitable" | {| class="wikitable" | ||
| - | ! | + | !Kai(100pg)||10x buffer KOD plus ver2||2M dNTP||MgSO4||Primer Mix(F&R)||Kod-plus ver2||MilliQ||total |
|- | |- | ||
| - | + | |0.5||2.5||2.5||1.5||1.5||0.5||16||25 | |
|} | |} | ||
{| class="wikitable" | {| class="wikitable" | ||
!PreDenature||Denature||Annealing||Extension||cycle | !PreDenature||Denature||Annealing||Extension||cycle | ||
|- | |- | ||
| - | | | + | |94°C||98°C||57°C||68°C||-- |
|- | |- | ||
| - | | | + | |2m||10s||30s||2m||30cycles |
|} | |} | ||
</div> | </div> | ||
<!-- ここまでをコピーしてね --> | <!-- ここまでをコピーしてね --> | ||
Revision as of 04:43, 24 September 2013
Contents |
Sep 9
PCR
| KaiA(plasmid) | 10x buffer | dNTPs | MgSO4 | Primer(661-RBS-KaiA-fwd) | Primer(kaiA-662-rev-ver2) | Kod-plus ver2 | MilliQ | total |
|---|---|---|---|---|---|---|---|---|
| 1.0 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 15.5 | 25 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 98°C | 58.5°C | 68°C | -- |
| 2min | 10s | 30s | 30s | 30cycles |
| KaiB(plasmid) | 10x buffer | dNTPs | MgSO4 | Primer(661-RBS-KaiA-fwd-ver2) | Primer(kaiB-663-rev-ver2) | Kod-plus ver2 | MilliQ | total |
|---|---|---|---|---|---|---|---|---|
| 1.0 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 15.5 | 25 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 98°C | 58.5°C | 68°C | -- |
| 2min | 10s | 30s | 30s | 30cycles |
| P-KaiBC(PCR products) | 10x buffer | dNTPs | MgSO4 | Primer(prefix-P-kaiBC-fwd-ver2) | Primer(P-KaiBC-suffix-rev-verw) | Kod-plus ver2 | MilliQ | total |
|---|---|---|---|---|---|---|---|---|
| 0.7 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 15.8 | 25 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 98°C | 58.5°C | 68°C | -- |
| 2min | 10s | 30s | 30s | 30cycles |
| DT(plasmid) | 10x buffer | dNTPs | MgSO4 | Primer(664-DT-fwd-ver2) | Primer(DT-suffix-660-rev-ver2) | Kod-plus ver2 | MilliQ | total |
|---|---|---|---|---|---|---|---|---|
| 1.0 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 15.5 | 25 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 98°C | 58.5°C | 68°C | -- |
| 2min | 10s | 30s | 30s | 30cycles |
Miniprep
| DNA | concentration[µg/mL] | 260/280 | 260/230 |
|---|---|---|---|
| 9/8 pSB1C3 | 83.2 | 1.45 | 0.76 |
| 9/8 RBS-lysis2-DT | 72.8 | 1.67 | 0.97 |
| 9/8 spinach-DT | 152.6 | 1.88 | 1.97 |
| Nuclease-Free water | -0.4 | -14.30 | 0.25 |
Restriction Enzyme Digestion
| DNA | restricion enzyme | restriction enzyme | Buffer | BSA | MilliQ | total | |
|---|---|---|---|---|---|---|---|
| 2 cuts | （°°） µL | µL | µL | µL | µL | µL | µL |
| NC | µL | µL | µL | µL | µL | µL | µL |
Transformation
| Name | Sample | Competent Cells | Total | Plate |
|---|---|---|---|---|
| いでんし | なんちゃらµL | ごにょごにょµL | ほにゃららµL | こーせーぶっしつ |
| いかどうよう | ||||
Ligation
| state | Vector | Inserter | Ligation High ver.2 | ||
|---|---|---|---|---|---|
| experiment | name | xx µg/mL | name | xx µg/mL | xx µg/mL |
| NC | name | xx µg/mL | name | xx µg/mL | xx µg/mL |
Colony PCR
| Sample | base pair |
|---|---|
| 9/8 Plac-spinach-DT-(1) | 659 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 94°C | 55°C | 68°C | -- |
| 5m | 30s | 30s | 40s | 30cycles |
| Sample | base pair |
|---|---|
| 9/8 Plac-spinach-DT-(2) | 659 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 94°C | 55°C | 68°C | -- |
| 5m | 30s | 30s | 40s | 30cycles |
| Sample | base pair |
|---|---|
| 9/8 Pcon-pt181atte-DT-(1) | 584 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 94°C | 55°C | 68°C | -- |
| 5m | 30s | 30s | 40s | 30cycles |
Gel Extraction
| Lane | DNA | Enzyme |
|---|---|---|
| 1 | 100bpladder | -- |
| 2 | Spinach-DT | X+P |
| 3 | ||
| 5 | RBS-lacZα-DT | X+P |
| 6 |
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
| Name | quantity | concentration[µg/mL] | 260/280 | 260/230 |
|---|---|---|---|---|
| （´ω｀） | ||||
| （ΦωΦ^） |
Ligation
| state | Vector | Inserter | Ligation High ver.2 | ||
|---|---|---|---|---|---|
| experiment | name | xx µg/mL | name | xx µg/mL | xx µg/mL |
| NC | name | xx µg/mL | name | xx µg/mL | xx µg/mL |
PCR
| Kai(100pg) | 10x buffer KOD plus ver2 | 2M dNTP | MgSO4 | Primer Mix(F&R) | Kod-plus ver2 | MilliQ | total |
|---|---|---|---|---|---|---|---|
| 0.5 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 16 | 25 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 98°C | 57°C | 68°C | -- |
| 2m | 10s | 30s | 2m | 30cycles |
