Difference between revisions of "Team:TUDelft/Wetlab/Protocols"

NOTE: All work is performed within a sterile field created by a bunsen burner flame.

1. For one cryostock, take a 1.5mL sample from an overnight liquid cultures.
2. Centrifuge the 2mL tubes at 2000rpm for 10 min.
3. Decant the supernatant without disturbing the pellet.
4. Add fresh sterile LB medium to the pellet, 1/3 volume of the starting volume of the culture.
5. Completely resuspended the pellet by vortexing the tube.
6. Add sterile 80% glycerol solution, the same volume as fresh LB in step 4.
7. Mix by vortexing.
8. Make a 1mL aliquot in cryotubes and label it with the cell type, plasmid type, protein type, operator and date.
9. Store the vials at -80ºC and update the inventory.

1. 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.
2. 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).
3. 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:

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

* NOTE: Use GoTaq Buffer Green when it is required to run a verification gel afterwards.
4. Pipette 45 µL of mix into each PCR tube (one tube per colony).
5. Centrifuge the colony mixture for 5 minutes at 16,000 x g.
6. Add 5 µL of supernatant of colony mixture to each PCR tube.
7. Put the tubes in the PCR machine and apply the following program (it needs to be adjusted for primers annealing temperature and extension time):



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

8. 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.

1. Decide on which enzyme(s) to cut with. Check online what buffer the enzyme(s) work(s) in (NEB). For most of the enzymes, the SmartCut buffer 10X can be used.
2. Prepare a sample a sample as follows:

Component	Volume (µL)
10x CutSmart buffer (NEB)	2
Fragment (~1-2 μg)	X (depending on the concentration)
Restriction Enzyme 1	1
Restriction Enzyme 2 (optional)	1
MilliQ	20 - (3 + X) add up to 20 µL

3. Incubate for 4 hours at 37 °C.
4. Inactivate the restriction enzyme(s) by heating to 65 °C for 10 minutes.
   DNA Purification (PCR) protocol for subsequent cloning strategies.