Template:Kyoto/Notebook/Sep 9
From 2013.igem.org
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+ | ===Gel Extraction=== | ||
+ | <!-- こっから --> | ||
+ | <div class="experiment"> | ||
+ | <span class="author">Yoshida</span> | ||
+ | {| class="wikitable" | ||
+ | !Lane||DNA||Enzyme | ||
+ | |- | ||
+ | |1||100bp ladder||-- | ||
+ | |- | ||
+ | |2||rowspan="2"|KaiA||rowspan="2"|-- | ||
+ | |- | ||
+ | |3 | ||
+ | |- | ||
+ | |4||rowspan="2"|KaiB||rowspan="2"|-- | ||
+ | |- | ||
+ | |5 | ||
+ | |- | ||
+ | |6||rowspan="2"|P-KaiBC||rowspan="2"|-- | ||
+ | |- | ||
+ | |7 | ||
+ | |- | ||
+ | |8||rowspan="2"|DT||rowspan="2"|-- | ||
+ | |- | ||
+ | |9 | ||
+ | |- | ||
+ | |10||NC||-- | ||
+ | |- | ||
+ | |11||100bp ladder||-- | ||
+ | |} | ||
+ | [[File:igku_xxbeforexx.xxx]]<br> | ||
+ | [[File:igku_xxafterxx.xxx]]<br> | ||
+ | {| class="wikitable" | ||
+ | !Name||concentration[µg/mL]||260/280||260/230 | ||
+ | |- | ||
+ | |(´ω`)|| || || | ||
+ | |- | ||
+ | |(ΦωΦ^)|| || || | ||
+ | |} | ||
+ | </div> | ||
+ | <!-- ここまでをコピーしてね --> | ||
+ | |||
===Miniprep=== | ===Miniprep=== | ||
<div class="experiment"> | <div class="experiment"> |
Revision as of 09:38, 24 September 2013
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 |
Gel Extraction
Lane | DNA | Enzyme |
---|---|---|
1 | 100bp ladder | -- |
2 | KaiA | -- |
3 | ||
4 | KaiB | -- |
5 | ||
6 | P-KaiBC | -- |
7 | ||
8 | DT | -- |
9 | ||
10 | NC | -- |
11 | 100bp ladder | -- |
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
Name | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|
(´ω`) | |||
(ΦωΦ^) |
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 |
Fe(Ⅲ)
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 |
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 |
Gel Extraction
Lane | DNA | Enzyme |
---|---|---|
1 | 100bp ladder | -- |
2 | Spinach-DT | XbaI+PstI |
3 | ||
5 | RBS-lacZα-DT | XbaI+PstI |
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 | 16.0 | 25.0 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94°C | 98°C | 57°C | 68°C | -- |
2m | 10s | 30s | 2m | 30cycles |
Kai(400pg) | 10x buffer KOD plus ver2 | 2M dNTP | MgSO4 | Primer Mix(F&R) | Kod-plus ver2 | MilliQ | total |
---|---|---|---|---|---|---|---|
2.0 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 14.5 | 25.0 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94°C | 98°C | 57°C | 68°C | -- |
2m | 10s | 30s | 2m | 30cycles |
Kai(800pg) | 10x buffer KOD plus ver2 | 2M dNTP | MgSO4 | Primer Mix(F&R) | Kod-plus ver2 | MilliQ | total |
---|---|---|---|---|---|---|---|
4.0 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 12.5 | 25.0 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94°C | 98°C | 57°C | 68°C | -- |
2m | 10s | 30s | 2m | 30cycles |
Plac(100pg) | 10x buffer KOD plus ver2 | 2M dNTP | MgSO4 | Primer Mix(F&R) | Kod-plus ver2 | MilliQ | total |
---|---|---|---|---|---|---|---|
0.3 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 16.2 | 25.0 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94°C | 98°C | 57°C | 68°C | -- |
2m | 10s | 30s | 2m | 30cycles |
Plac(200pg) | 10x buffer KOD plus ver2 | 2M dNTP | MgSO4 | Primer Mix(F&R) | Kod-plus ver2 | MilliQ | total |
---|---|---|---|---|---|---|---|
0.6 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 15.9 | 25.0 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94°C | 98°C | 57°C | 68°C | -- |
2m | 10s | 30s | 2m | 30cycles |
Plac(400pg) | 10x buffer KOD plus ver2 | 2M dNTP | MgSO4 | Primer Mix(F&R) | Kod-plus ver2 | MilliQ | total |
---|---|---|---|---|---|---|---|
1.2 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 15.3 | 25.0 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94°C | 98°C | 57°C | 68°C | -- |
2m | 10s | 30s | 2m | 30cycles |
Plac(800pg) | 10x buffer KOD plus ver2 | 2M dNTP | MgSO4 | Primer Mix(F&R) | Kod-plus ver2 | MilliQ | total |
---|---|---|---|---|---|---|---|
2.4 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 14.1 | 25.0 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94°C | 98°C | 57°C | 68°C | -- |
2m | 10s | 30s | 2m | 30cycles |
Plac(nega) | 10x buffer KOD plus ver2 | 2M dNTP | MgSO4 | Primer Mix(F&R) | Kod-plus ver2 | MilliQ | total |
---|---|---|---|---|---|---|---|
0.0 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 16.5 | 25.0 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94°C | 98°C | 57°C | 68°C | -- |
2m | 10s | 30s | 2m | 30cycles |
Liquid Culture
Sample | medium |
---|---|
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 |
PCR
KaiA(KaiABC) 100pg/µL | 10x buffer KOD plus ver2 | dNTPs | MgSO4 | Primer Mix(Fwd&Rev) | KOD plus ver2 | MilliQ | total |
---|---|---|---|---|---|---|---|
2.0 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 14.5 | 25 |
4.0 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 12.5 | 25 |
8.0 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 8.5 | 25 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94°C | 98°C | 60°C | 68°C | -- |
2m | 10s | 30s | 1m | 30cycles |
PKaiBC 400pg/µL | 10x buffer KOD plus ver2 | dNTPs | MgSO4 | Primer Mix(Fwd&Rev) | KOD plus ver2 | MilliQ | total |
---|---|---|---|---|---|---|---|
0.5 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 16.0 | 25 |
1.0 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 15.5 | 25 |
2.0 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 14.5 | 25 |
PreDenature | Denature | Annealing | Extension | cycle |
---|---|---|---|---|
94°C | 98°C | 60°C | 68°C | -- |
2m | 10s | 30s | 1m | 30cycles |
Liquid Culture
Sample | medium |
---|---|
9/2 Spinach-DT | Plusgrow medium(+CP) |
8/29 Plux-RBS-GFP-DT | Plusgrow medium |
9/6 master plate | Plusgrow medium(+Amp) |
3 Pcon-PT181 attenuator | Plusgrow medium |
Gel Extraction
Lane | DNA | Enzyme |
---|---|---|
1 | 1kbp ladder | -- |
3 | lysis1 | XbaI+PstI |
4 | ||
5 | ||
6 |
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
Name | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|
(´ω`) | |||
(ΦωΦ^) |
Lane | DNA | Enzyme |
---|---|---|
1 | 1kbp ladder | -- |
3 | lysis3 | XbaI+PstI |
4 | ||
5 | ||
6 |
File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx
Name | concentration[µg/mL] | 260/280 | 260/230 |
---|---|---|---|
(´ω`) | |||
(ΦωΦ^) |
Electrophoresis
Lane | Sample | Enzyme1 | Enzyme2 |
---|---|---|---|
1 | 1kb ladder | -- | -- |
2 | Plac 100pg | -- | -- |
3 | Plac 200pg | -- | -- |
4 | Plac 400pg | -- | -- |
5 | Plac 800pg | -- | -- |
6 | NC | -- | -- |
7 | 1kb ladder | -- | -- |