Difference between revisions of "Team:TUDelft/Wetlab/Protocols"
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Revision as of 09:09, 17 September 2018
Text to write to introduce the protocols
- Under sterile conditions, pick a colony and dilute it in 10 µL of milli-Q water.
NOTE: After you pick the colony, it cannot be used again. It is therefore recommended to make a 'back-up'-plate where you grow the colonies again. This plate should be incubated overnight at 37 °C. - Incubate at 90 °C for 10 min.
NOTE: Instead of separate 'cooking' of the cells before the PCR, this step can be incorporated in the PCR program. The initial denaturation step at 98 °C should then be prolonged to 5 minutes (300 seconds). - Prepare the GoTaq master mix for all the samples in a single 1.5 mL tube.
NOTE: mind pipetting errors so prepare at little bit more master mix!
For one sample: - Pipette 45 µL of mix into each PCR tube (one tube per colony).
- Centrifuge the colony mixture for 5 minutes at 16,000 x g.
- Add 5 µL of supernatant of colony mixture to each PCR tube.
- Put the tubes in the PCR machine and apply the following program (it needs to be adjusted for primers annealing temperature and extension time):
- The PCR product(s) can be checked on gel. In order to do so, cast a gel and prepare the samples according to the DNA electrophoresis protocol.
| Component | Volume (µL) |
|---|---|
| GoTaq 5x buffer | 10 |
| 10 mM dNTPs | 1 |
| Primer forward (10µM) | 1 |
| Primer reverse (10µM) | 1 |
| Sterile milli-Q | 31.8 |
| Gotaq polymerase (5u/µL) | 0.2 |
| Total | 45 |
| Step | Time (s) | Temperature (°C) |
|---|---|---|
| Initial denaturation | 150 | 98 |
| Denaturation | 60 | 94 |
| Annealing | 60 | 60 (depending on primers) |
| Extension | 60 sec per kb DNA | 72 |
| Final extension | 600 | 72 |
| Hold | ∞ | 4 |
- Under sterile conditions, pick a colony and dilute it in 10 µL of milli-Q water.
NOTE: After you pick the colony, it cannot be used again. It is therefore recommended to make a 'back-up'-plate where you grow the colonies again. This plate should be incubated overnight at 37 °C. - Incubate at 90 °C for 10 min.
NOTE: Instead of separate 'cooking' of the cells before the PCR, this step can be incorporated in the PCR program. The initial denaturation step at 98 °C should then be prolonged to 5 minutes (300 seconds). - Prepare the GoTaq master mix for all the samples in a single 1.5 mL tube.
NOTE: mind pipetting errors so prepare at little bit more master mix!
For one sample: - Pipette 45 µL of mix into each PCR tube (one tube per colony).
- Centrifuge the colony mixture for 5 minutes at 16,000 x g.
- Add 5 µL of supernatant of colony mixture to each PCR tube.
- Put the tubes in the PCR machine and apply the following program (it needs to be adjusted for primers annealing temperature and extension time):
- The PCR product(s) can be checked on gel. In order to do so, cast a gel and prepare the samples according to the DNA electrophoresis protocol.
| Component | Volume (µL) |
|---|---|
| GoTaq 5x buffer | 10 |
| 10 mM dNTPs | 1 |
| Primer forward (10µM) | 1 |
| Primer reverse (10µM) | 1 |
| Sterile milli-Q | 31.8 |
| Gotaq polymerase (5u/µL) | 0.2 |
| Total | 45 |
| Step | Time (s) | Temperature (°C) |
|---|---|---|
| Initial denaturation | 150 | 98 |
| Denaturation | 60 | 94 |
| Annealing | 60 | 60 (depending on primers) |
| Extension | 60 sec per kb DNA | 72 |
| Final extension | 600 | 72 |
| Hold | ∞ | 4 |