"
Template:Kyoto/Notebook/Sep 11
From 2013.igem.org
(Difference between revisions)
(→Restriction Enzyme Digestion) |
(→Restriction Enzyme Digestion) |
||
| Line 119: | Line 119: | ||
! ||DNA||E||X||S||P||buffer||BSA||MilliQ||Total|| | ! ||DNA||E||X||S||P||buffer||BSA||MilliQ||Total|| | ||
|- | |- | ||
| - | |pSB4K5( | + | |pSB4K5(EcoR1+S)||9.4µL|| 1µL|| 0µL|| 1µL|| 0µL|| 3µL|| 3µL|| 12.6µL||30 µL |
|- | |- | ||
|pSB4K5(E+S) negative||0.5µL|| 0µL|| 0µL|| 0µL|| 0µL|| 1µL|| 1µL|| 7.5µL|| 10µL | |pSB4K5(E+S) negative||0.5µL|| 0µL|| 0µL|| 0µL|| 0µL|| 1µL|| 1µL|| 7.5µL|| 10µL | ||
Revision as of 13:19, 24 September 2013
Contents |
Sep 11
PCR
| pKaiBC(200p) | 10*buffer KOD plus | dNTPs | MgSO4 | primer mix(F&R) | KOD plus | 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 | 60°C | 68°C | 30 |
| 2min | 10sec | 30sec | 30sec |
| pKaiBC(400p) | 10*buffer KOD plus | dNTPs | MgSO4 | primer mix(F&R) | KOD plus | MilliQ | total |
|---|---|---|---|---|---|---|---|
| 1 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 15.5 | 25 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 98°C | 60°C | 68°C | 30 |
| 2min | 10sec | 30sec | 30sec |
| pKaiBC(800p) | 10*buffer KOD plus | dNTPs | MgSO4 | primer mix(F&R) | KOD plus | MilliQ | total |
|---|---|---|---|---|---|---|---|
| 2 | 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 | 30 |
| 2min | 10sec | 30sec | 30sec |
| pKaiBC(1ng) | 10*buffer KOD plus | dNTPs | MgSO4 | primer mix(F&R) | KOD plus | MilliQ | total |
|---|---|---|---|---|---|---|---|
| 2.5 | 2.5 | 2.5 | 1.5 | 1.5 | 0.5 | 14 | 25 |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 98°C | 60°C | 68°C | 30 |
| 2min | 10sec | 30sec | 30sec |
colony PCR
| Sample | base pair |
|---|---|
| 9/9Pcon-lacZα(p3B4k5) | 712bp |
| 9/9Pcon-spinach-DT(p3B4k5) | 605bp |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 42s |
| Sample | base pair |
|---|---|
| Pbad\araC-RBS-luxI-DT |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 94°C | 55°C | 68°C | -- |
| 5min | 30s | 30s | 2min |
| Sample | base pair |
|---|---|
| 9/9Pcon+RBS-luxI-DT | 1079bp |
| 9/10Pcon+sptamer12_1R-DT | 584bp |
| 9/10DT+Pcon-pT181 attenuater 1 | 781bp |
| 9/10Fusion1 attenuater(p3B1C3)-1 | 638bp |
| PreDenature | Denature | Annealing | Extension | cycle |
|---|---|---|---|---|
| 94°C | 94°C | 55°C | 68°C | -- |
| 50sec | 30sec | 30sec | 1min |
Restriction Enzyme Digestion
| DNA | E | X | S | P | buffer | BSA | MilliQ | Total | ||
|---|---|---|---|---|---|---|---|---|---|---|
| pSB4K5(EcoR1+S) | 9.4µL | 1µL | 0µL | 1µL | 0µL | 3µL | 3µL | 12.6µL | 30 µL | |
| pSB4K5(E+S) negative | 0.5µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL | |
| RBS-lacZα-DT(E+S) | 10µL | 1µL | 0µL | 1µL | 0µL | 3µL | 3µL | 12µL | 30µL | |
| RBS-lacZα-DT(E+S) negative | 0.5µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL | |
| Ptet-pT181 antisense(S+P) | 10.8µL | 0µL | 0µL | 1µL | 1µL | 3µL | 3µL | 12µL | 30µL | |
| Ptet-pT181 antisense(S+P) negative | 0.5µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.5µL | 10µL | |
| Pcon-RBS-lacZα-DT(E+S) | 7.1µL | 1µL | 0µL | 1µL | 0µL | 3µL | 3µL | 14.9µL | 30µL | |
| Pcon-RBS-lacZα-DT(E+S) negative | 0.4µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 7.6µL | 10µL | |
| RBS-lysis2-DT(X+P) | 22µL | 0µL | 1µL | 0µL | 1µL | 3µL | 3µL | 0µL | 30µL | |
| RBS-lysis2-DT(X+P) negative | 1.4µL | 0µL | 0µL | 0µL | 0µL | 1µL | 1µL | 6.6µL | 10µL |
