Team:UCopenhagen/Experiments

PROTOCOLS

PCR Cleanup

Reagents required:
• PCR clean up kit (E.N.Z.A)
• PCR reaction product

Procedure:
1.) Transfer 30-50 μL of the PCR product to a new 1.5 mL microcentrifuge tube.
2.) Add 4-5 times volume of the CP buffer.
3.) Briefly vortex and centrifuge the samples.
4.) Insert the Hibind DNA mini columns into the 2 mL collection tubes.
5.) Add the samples from step 3 to the Hibind DNA mini column.
6.) Centrifuge at 13000 g for 60 seconds at room temperature. Discard the filtrate and re use the collection tubes.
7.) Add 700 μL of DNA wash buffer diluted with ethanol to the tubes.
8.) Centrifuge at maximum speed for 60 seconds. Discard the filtrate and re use the collection tubes.
9.) Repeat step 7 for a second DNA wash buffer step.
10.) Centrifuge the empty Hibind DNA columns at maximum speed for 2 minutes to dry the column. This step is critical to remove the trace ethanol.
11.) Transfer the Hibind DNA column to a new 1.5 mL microcentrifuge tube.
12.) Add 50 μL of the elution buffer directly into the Hibind DNA column.
13.) Keep at room temperature for 2 minutes and centrifuge at maximum speed for 60 seconds.
14.) Store the DNA at -20℃.

Plasmid DNA Extraction

Reagents required:
• E.N.Z.A Plasmid DNA mini prep kit
• Culture

Procedure:
1.) Vortex the liquid cultures and use 1-5 mL for the plasmid extraction.
2.) Centrifuge at 10000 g for 1 min at room temperature. Discard the pellet.
3.) Add 250 μL of solution I, vortex the samples and transfer the suspension to a new microcentrifuge tube.
4.) Add 250 μL of solution II, invert the samples and wait for 2-3 minutes.
5.) Add 350 μL Solution III and invert the tube several times.
6.) Centrifuge the samples at maximum speed for 10 minutes.
7.) Place the Hibind DNA columns onto the 2 mL collection tubes.
8.) Transfer the supernatant from step 6 into the Hibind DNA columns.
9.) Centrifuge at maximum speed for 1 minute and discarded the filtrate.
10.) Add 500 μL of HBC buffer and centrifuge it at maximum speed for 1 minute and discard the supernatant.
11.) Add 700 μL DNA Wash Buffer and centrifuge it at maximum speed for 30 seconds and discard the supernatant.
12.) Repeat step 11 for a second DNA wash step.
13.) Centrifuge the empty Hibind DNA column for 2 min at maximum speed to dry the column.This will remove trace amounts of ethanol.
14.) Transfer the Hibind DNA column into a new microcentrifuge tube.
15.) Add 50 μL of Elution Buffer, let it sit at room temperature for 2 minutes and centrifuge at maximum speed for 1 minute.
16.) Store DNA at -20℃.

NanoDrop DNA quantification

Reagents required:
• DNA samples
• Milli-Q water
• Nanodrop device
• Pipette

Procedure:
1.) Clean the measurement surface with a piece of tissue and ethanol.
2.) Use 1 μL of Milli-Q water as a blank.
3.) Clean the measurement surface with a piece of tissue.
4.) Use 1 μL of sample to measure the concentration.
NOTE: It is best to measure the same sample in triplo and use the average value. If you have multiple samples, clean the measurement surface in between measurements.
5.) When done measuring, wipe the measuring surface with a tissue and Milli-Q water.

USER Cassette linearization reaction

Reagents required:
• Purified PCR product
• Restriction enzyme NotI HF
• Milli-Q water
• 10X cut smart buffer

Procedure:
1.) Prepare sample in a 0.5 mL microcentrifuge tube as follows.

Component	Volume (μL)
10X cut smart buffer	4
Purified PCR product	30
Restriction enzyme Dpn1	1
Milli-Q	5

2.) Incubate for 1.5 hours at 37℃.
3.) Heat inactivate the enzyme by heating it at 80℃ for 20 minutes.

Yeast Colony PCR

Reagents required:
• NaOH solution
• MangoMix master mix solution
• F1-primer
• F2-primer
• R1-primer
• Template: Colonies from overnight plates

Procedure:

Yeast cell lysis:
1.) Calculate the number of colonies and mark required number of PCR tubes.
2.) Add 20 μL of 20mM NaoH to all the tubes.
3.) Pick selected colonies using a pipette tip and inoculate into the tubes containing NaoH.
4.) Place the tubes in the PCR machine and run it for 10 minutes at 99℃.

PCR reaction:
1.) Use a commercial mastermix called MangoMix. This mastermix solution contains taq polymerase and dNTPs.
2.) Take new PCR tubes and add 5 μL of Mango master mix, 0.5 μL of each primers and 3.5 μL of water to a total volume of 9 μL.
3.) Add 1 μL of the template to each of the respective tubes.
4.) Place the tubes in the PCR machine and run a programme with following steps:

30-35 cycles for steps marked in red
96 degree celsius: 30 seconds

96 degree celsius: 30 seconds
55 degree celsius: 25 seconds
72 degree celsius: 2 minutes and 30 seconds

72 degree celsius: 5 minutes
12 degree celsius: Infinity

Bacterial Colony PCR

Reagents required:
• 10X X7 PCR buffer
• dNTP
• F-primers (10 μM)
• R-primers (10 μM)
• Template
• X7-Polymerase
• mQ Water

Procedure:
1.) Selected isolated colonies from the plate and using a sterile cocktail stick, remove a small quantity (such that it is visible on the end of the stick) of cells from the plate. This will serve as the template in the PCR mix.
2.) Label the PCR tubes with a number of g-blocks to be amplified.
3.) Calculate the number of samples and prepare a master mix (all the PCR reagents except the template).
4.) Transfer the master mix to the labelled PCR tubes and add the necessary templates to all the tubes.
5.) Set the PCR machine - Calculate the annealing temperature and extension time (20-30s/Kb)
6.) Place all the PCR tubes in the machine and run the PCR with the pre-set programme.

Competent Bacterial Cells

Reagents required:
• LB agar plates
• LB liquid media
• E.coli (WT)
• Eppendorfs
• TSS buffer

Procedure:
1.) From the overnight culture, add 1 mL of E.coli to 99 mL of fresh LB media and grow to OD600 to 0.4-0.5.
2.) Place the eppendorfs on ice and make sure that TSS buffer is chilled in the fridge (4℃).
3.) Split the culture into different falcon tubes and incubate them on ice for 10 minutes.
4.) Centrifuge the falcon tubes for 10 minutes at 3000 RPM and 4℃ ( The centrifuge needs to be cooled down to 4℃ before use).
5.) Discard the supernatant and resuspend the pellet in TSS buffer.
6.) Make aliquots into cooled eppendorf tubes and freeze them in -80℃.

Standard PCR

Reagents required:
• 10X X7 PCR buffer
• dNTP
• F-primers (10 μM)
• R-primers (10 μM)
• Template
• X7-Polymerase
• mQ Water

Procedure:
1.) Label the PCR tubes with number of g-blocks to be amplified.
2.) Calculate the number of samples and prepare a master mix (all the PCR reagents except the template).
3.) Transfer the master mix to the labelled PCR tubes and add the necessary templates to all the tubes.
4.) Set the PCR machine - Calculate the annealing temperature and extension time (20-30s/Kb).
5.) Place all the PCR tubes in the machine and run the PCR with the pre-set programme.

Dilution of G-blocks and primers

Reagents required:
• Primers
• g-blocks
• mQ water

Procedure:
1.) Dilute the primers and g-blocks in the tubes they came in, by making the solutions as following:
2.) Add 100 μL of Milli-Q water to the g-block fragments (1000ng) to make it 10 ng/μL .
3.) For the primers, make up to 100 µM concentrations.

DNA transformation in E. Coli

Reagents required:
• Competent cells
• Vector containing DNA

Procedure:
1.) Add 1% volume of the vector to competent cells.
2.) Mix by gently flicking the tube. Vortex will prevent transformation.
3.) Incubate the cells on ice for 30-60 minutes.
4.) Water bath at 42℃ for 45 seconds.
5.) Again incubate on ice for 3-5 minutes.
6.) Add 950 μL of LB and place them in shaking incubator at 37℃ for 1 hour.
7.) Centrifuge at 13000 RPM for 1 minute.
8.) Discard the supernatant slowly, leaving behind 50 μL-100 μL in the tube.
9.) Resuspend the pellet using a pipette and spread on to an appropriate selection plate and incubate overnight at 37℃.

Agarose Gel Electrophoresis

Reagents required:
• 1% agarose
• Gel red
• Electrophoretic chamber
• Samples to be loaded
• Molecular weight marker (DNA ladder)

Procedure:
1.) For a 1% (w/v) gel in TAE, weigh agarose powder for a 1% (w/v) 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.
3.) Pour gel in gel tray and mix well with Gel Red. For a small gel (~ 40 mL solution) add 1 μL of Gel red; for a large gel (~80 mL) add 2 µL of Gel red. Add a comb to create wells for the samples. Allow the agarose to solidify.
4.) Transfer the gel to the electrophoresis cell, remove the combs and cover the gel in TAE buffer (1X).
5.) Prepare the electrophoresis samples by adding Nucleic Acid Loading Buffer conform the manufacturer’s instructions.
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.
7.) Connect the cables of the gel tray following the colour code and run at 80-110V for 40-60 min.

Preparation of agar plates

Reagents required:
• LB agar
• Milli-Q water
• Petri dishes

Procedure:
1.) Dissolve X grams of LB-agar in X mL of Milli-Q.
2.) Autoclave the LB-agar solution.
3.) Cool the agar down to room temperature.
4.) Add the correct amount of the correct antibiotic. (We used 2 μL of antibiotic per mL of LB agar)
5.) Under sterile conditions, pour the LB-agar in empty Petri dishes (20-25 mL per plate).
6.) Leave the Petri dishes in the sterile bench with the lid partially off for approximately 5 minutes so the agar can solidify.

Restriction digestion assay

Reagents required:
• 10X cut smart buffer
• Restriction enzymes
• DNA sample

Procedure:
1.) Decide on which restriction enzyme(s) to cut with. Find out what buffer the enzyme(s) work(s) in. In general, SmartCut buffer can be used.
2.) Prepare the sample as follows:

Component	Volume (μL)
DNA	(~1 µg is required)
Buffer (CutSmart)	2
Restriction enzyme(s) (5u/µg)	1 µL each
Milli-Q	Up to 20-25 µL

2.) Incubate for one hour at 37 °C.
3.) Inactivate the restriction enzyme(s) by heating to 65 °C for 10 minutes.
NOTE: This last step can be skipped if the sample is evaluated on gel immediately after.

Raffinose media

Reagents required:
• Raffinose
• Yeast nitrogen base
• Synthetic drop out media
• Amino acids
• NaOH

Procedure:
1.) Add all the necessary reagents in necessary quantities.
2.) The quantities used to prepare 1L of the media are given below:
Raffinose - 20g
Yeast nitrogenous base - 6.7g
Synthetic drop out media - 1.4g
Amino acids:
0.04g Trp, 0.04g Leu, 0.02 His
3.) Make up the volume to 1 L.
4.) Adjust the pH(5.8-6.2) using 10% NaOH.

USER Ligation

This experiment is carried out to ligate the gene of interest with its promoter and vector backbone.
Note: The Gene of interests used in this experiment have already been amplified with primers containing USER sequence.

Reagents required:
• USER enzyme
• Cut smart buffer
• Promoter
• Gene of interest
• Vector backbone
• Milli-Q water

Procedure:
1.) Create a master mix using Milli-Q water, USER enzyme and cutsmart buffer as shown below.
2.) For each ligation, add the gene fragments, promoters and vector backbones separately.
3.) Add 7 μL of the master mix to each tube. Finally, place the samples in the PCR and set the following conditions:
37 °C for 30 min
25 °C for 15 min
20 °C for 10 min
12 °C indefinitely

Compound	Volume (μL)
USER enzyme	1
CutSmart buffer	1
Promoter	1
Gene	1
Vector backbone	1
Vector backbone	1
Milli-Q	5
Total	10

2.) Incubate for 1.5 hours at 37℃.
3.) Heat inactivate the enzyme by heating it at 80℃ for 20 minutes.

Yeast transformation

Reagents required:

Reagents required:
• O/N yeast culture
• YPD media
• Cuvettes
• Salmon sperm
• Lithium acetate
• Vector backbones
• Milli-Q

Procedure:
1.) Mix 0.9 mL YPD media and 0.1 mL of overnight culture in a cuvette and measure OD at 600nm.
2.) Dilute the culture to an OD(600nm) = 0.25 based on the OD value measured.
3.) Incubate the culture for another 4h to reach an OD(600nm) of approximately 1.
4.) Boil the ssDNA(Salmon sperm) for 10 mins at 99°C in the heat block and keep it on ice.
5.) Spin down the culture in the falcon tube at 3000RCF for 10 mins at room temperature.
6.) Pour the supernatant into a waste tube and resuspend in 1mL of LiAc.
7.) Transfer the culture into 1.5 mL of Eppendorf tubes and spin down at 7000 RCF for 1 min at room temperature.
8.) Remove the supernatant carefully with a pipette and resuspend the pellet in a 100 μL of 0.1M LiAc.
9.) Add 30 μL of salmon sperm and mix everything gently.
10.) Add 1 mL of PLI and mix well.
11.) Transfer the DNA samples into 1.5 mL eppendorf tubes and 200 μL of the yeast culture.
12.) Heat shock the samples for 30 mins at 42 °C in a heat block.
13.) After transformation, spin down the cells at 7000RCF for 1 min at room temperature and discard the supernatant.
14.) Resuspend the pellet in 1 mL of water.
15.) Spin down the cells at 8000RCF for 1 min at room temperature.
16.) Remove 900 μL of the water and resuspend the cells in the remaining water.
17.) Finally, add the transformed cells onto the necessary plates and spread it uniformly.
18.) Incubate the plates for 3 nights at 30°C.

Interleukine Induction Assay (200 µL, 96-well)

Reagents required:
• Cell cultures of the strains to be measured
• Growth media
• Dilution series of receptor ligands
• YeastBuster
• Luciferase substrate
• Sterile 96-well plate
• black 96-well plate

Procedure:
Incubation - keep sterile!
1.) Grow cells to desired OD600 in falcon tubes with YPD
2.) Optional: Spin down cells for 1 min. at 5000 RCF and discard supernatant
3.) Resuspend OR dilute cells in desired growth medium to desired concentration
4.) Transfer cells to sterile 96-well plate
5.) Add desired volume of biomarker dilution series to the wells (remember to note down what is in each well)
6.) Optional: Wrap plate in parafilm to reduce evaporation.
7.) Incubate in shaker at 30°C for desired incubation time

Luciferase assay 8.) Transfer ¾ of desired measurement volume to black 96-well plate
9.) Add ¼ of desired measurement volume YeastBuster with 1% luciferase substrate to each well
10.) Immediately measure luminescence from top with plate reader.