From 2013.igem.org
(Difference between revisions)
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| ===Miniprep=== | | ===Miniprep=== |
| <div class="experiment"> | | <div class="experiment"> |
- | <span class="author">Yoshida</span> | + | <span class="author">No name</span> |
| {|class="wikitable" | | {|class="wikitable" |
| !DNA||concentration[µg/mL]||260/280||260/230 | | !DNA||concentration[µg/mL]||260/280||260/230 |
Revision as of 09:39, 24 September 2013
Sep 9
PCR
Nakagawa
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 | --
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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
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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
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0.7 | 2.5 | 2.5 | 1.5 | 0.75 | 0.75 | 0.5 | 15.8 | 25
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PreDenature | Denature | Annealing | Extension | cycle
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94°C | 98°C | 58.5°C | 68°C | --
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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
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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
No name
DNA | concentration[µg/mL] | 260/280 | 260/230
|
9/8 pSB1C3 | 83.2 | 1.45 | 0.76
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9/8 RBS-lysis2-DT | 72.8 | 1.67 | 0.97
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9/8 spinach-DT | 152.6 | 1.88 | 1.97
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Nuclease-Free water | -0.4 | -14.30 | 0.25
|
Restriction Enzyme Digestion
じっけんしゃ
| 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
じっけんしゃ
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
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いかどうよう | | | |
|
| | | |
|
Ligation
No Name
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)
Shimizu & Hirano
Colony PCR
No name
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
じっけんしゃ
| 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
|
Ligation
じっけんしゃ
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
Tatsui and Yoshida
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
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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
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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
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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
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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
Restriction Enzyme Digestion
じっけんしゃ
| 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
Yoshida
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
|
Yoshida
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
No name
Sample | medium
|
9/2 Spinach-DT | Plusgrow medium(+CP)
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8/29 Plux-RBS-GFP-DT | Plusgrow medium
|
9/6 master plate | Plusgrow medium(+Amp)
|
3 Pcon-PT181 attenuator | Plusgrow medium
|
Electrophoresis
Yoshida
Lane | Sample | Enzyme1 | Enzyme2
|
1 | 1kb ladder | -- | --
|
2 | Plac 100pg | -- | --
|
3 | Plac 200pg | -- | --
|
4 | Plac 400pg | -- | --
|
5 | Plac 800pg | -- | --
|
6 | NC | -- | --
|
7 | 1kb ladder | -- | --
|
File:Igku xxxxxx.xxx