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

This protocol is based on the Pierce BCA protein assay kit by Thermo Scientific protocol.

1. Prepare a set of protein standards using one 2mg/mL Albumin Standard (BSA) ampule according to the table below: NOTE: Use the same diluent as the samples. The expected working range = 20-2000µg/mL.
   

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

2. Determine the amount of total volume of working reagent (WR) required by using the the following formula:
   Total volume WR = (# standards + # unknowns) × (# replicates) × (200 µl)
3. Prepare the BCA working reagent by mixing 50 parts of BCA Reagent A with 1 part of BCA Reagent B (50:1, Reagent A:B). NOTE: The WR is stable for several days when stored in a closed container at room temperature (RT).
4. Pipette 25µL of each standard or unknown sample replicate into a microplate well.
5. Add 200µL of the WR to each well and mix plate thoroughly.
6. Cover plate and incubate at 37°C for 30 minutes.
7. Cool plate to room temperature.
8. Measure the absorbance at or near 562nm on a plate reader.

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.