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.
   

Vial	Volume of MilliQ (µL)	Source of BSA	Volume of source BSA (µL)	Final BSA concentration (µg/µL)
A	0	Stock	300	2000
B	125	Stock	375	1500
C	325	Stock	325	1000
D	175	Vial B	175	750
E	325	Vial C	325	500
F	325	Vial E	325	250
G	325	Vial F/td>	325	125
H	400	Vial G	100	25
I	400	n/a/td>	0	0

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.

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.

This protocol spans over 3 days of execution time, starting from a -80°C mother stock. Throughout the protocol, it is recommended to work under aseptic conditions in order to prevent contamination risks.

Day 1

1. Keep the -80°C strain stock of interest on ice.
2. Streak the strain on solid selective medium and incubate overnight at 37 °C while shaking.

Day 2

1. Prepare a 10mL liquid starter culture with one of the colonies that grew on the selective plate. Let the culture grow overnight at 37°C, shaking at 180rpm.
2. Sterilise solutions of CaCl₂ 100 mM and of CaCl₂ 100 mM + 15 % Glycerol in advance.
   NOTE: Volumes depend on the total culture volume to be prepared in step 5. For example, if 1 L is used in step 7, 300 mL of CaCl₂ 100 mM and 10 mL of CaCl₂ 100 mM + 15 % glycerol will be used.

Day 3

1. Inoculate 1:100 of overnight culture in the desired volume of LB with antibiotic (eg. 10 mL of culture in 1 L of LB).
2. Incubate in a shaker at 37 °C, 180 rpm to OD600nm ~0.4-0.6 (measure OD600nm every 30 minutes).
3. Harvests the cells by centrifugation at 4000 x g for 5 minutes in centrifuge tubes, decant supernatant.
4. Resuspend cells by gently pipetting 1/5 (of the volume of LB from step 5) of ice-cold 100 mM CaCl₂ and incubate on ice for 20 minutes.
5. Pellet the cells by centrifugation at 4000 x g for 5 minutes in centrifuge tubes, decant supernatant.
6. Resuspend cells by gently pipetting 1/10 (of the volume of LB from step 5) of ice-cold 100 mM CaCl₂ and incubate on ice for 60 minutes.
7. Pellet the cells by centrifugation at 4000 x g for 5 minutes in centrifuge tubes, decant supernatant.
8. Resuspend cells by gently pipetting 1/100 (of the volume of LB from step 5) of ice-cold 100 mM CaCl₂ + 15% glycerol and keep on ice.
9. Chemical competent cells can either immediately be used for heat shock transformation , or stored in aliquots of 50 uL in microcentrifuge tubes at -80 °C.

1. Under aseptic conditions, pick a colony, resuspend it in 10 µL of milli-Q water.
   NOTE: A picked colony cannot be used again; it is recommended to restreak on a 'back-up'-plate and incubate it overnight at 37 °C.
2. Incubate the resuspended colony at 90 °C for 10 min. Spin the suspension down and use the supernatant as template DNA for the PCR.
   NOTE: Instead of separate boiling prior to PCR, this step can be incorporated in the PCR program. The initial denaturation step at 98 °C should then be prolonged to 5 minutes.
3. Make sure every PCR reaction is composed as follows:
   

   Component	Volume (µL)	Final concentration
   GoTaq 5x buffer*	10	1 X
   10 mM dNTPs	1	200 µM
   Primer forward (10µM)	1	200 nM
   Primer reverse (10µM)	1	200 nM
   Boiled colony supernatant	5
   Gotaq polymerase (5u/µL)	0.2	20 U/mL
   MilliQ	31.8

   * NOTE: Use GoTaq Buffer Green when it is required to run a verification gel afterwards.
4. Add 5 µL of supernatant of colony mixture to each PCR tube.
5. Close all tubes thoroughly and place them in a thermocycler with the following protocol:
   

   Step	Time (s)	Temperature (°C)
   Initial denaturation	150	98
   Denaturation	60	94
   Annealing	60	Tann*
   Extension	60 sec per kb DNA	72
   Final extension	600	72
   Hold	∞	4

   *NOTE: The annealing temperature (T_ann) is dependent on the melting temperature (T_m) of the primers used. It is recommended to have T_ann = T_m - 5°C.
6. 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.

Gel Electrophoresis and DNA staining makes use of mutagenic chemicals like EtBr, SYBR Safe or any other DNA staining. Wear protection (gloves) when carrying out this protocol, work in an assigned area for this work to prevent contamination of the rest of the lab.

1. Weigh agarose powder for a 0.8% (w/v) gel in TAE.
   E.g. weigh 1.6g and add to 200mL TAE buffer for a 0.8% gel.
2. Add weighed agarose in TAE buffer (1X) and warm the solution (in a microwave) until it is completely dissolved. Let the solution cool down to hand warm temperature.
   NOTE: Make sure the lid is not completely closed to avoid possible exploding of the glass bottle.
3. Pour gel in gel tray and mix well with SYBR Safe. For a small gel (~ 40 mL solution) add 1 µL of SYBR Safe; for a large gel (~80 mL) add 2 µL of SYBR Safe. Add a comb to create wells for the samples. Allow the agarose to solidify (approximately 20 minutes).
4. Transfer the gel to the electrophoresis cell, remove the combs and cover the gel in TAE buffer (1X).
   NOTE: mind the direction of DNA migration when placing the gel in the cell.
5. Prepare the electrophoresis samples by adding Nucleic Acid Loading Buffer conform the manufacturer’s directions.
6. Load the molecular weight marker (DNA ladder) in the first well according to manufacturer’s instructions (generally 3-5 µL) and load 5-10 µL of the dyed samples in the other wells.
   NOTE: Do not contaminate the loading buffer and ladder with SYBR Safe! Do not touch it while wearing a glove.
7. Connect the cables of the gel tray following the colour code and run at 80-110V for 40-60 min.
   NOTE: mind the direction of DNA migration when placing the lid on the cell.
   NOTE: time and voltage depend on the density of the gel and the length of the lane in the gel.

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.