Team:SDU-Denmark/Experiments

Experiments and protocols

"All great experiments are build upon a 1000 failed ones."

Experiments

To get and follow our results through the iGEM experience in the wet lab we used a range of different experiments. A collection of which experiments have been used and for what can be found here:
Assembling the DNA sequences of Cas13 and Cas12 and inserting it into an iGEM plasmid:
  1. PCR amplification and PCR overhang ligation
  2. DNA digestion and ligation
  3. Transformation
  4. Colony PCR

Protein expression and purification:
  1. Protein expression inducement
  2. Protein purification
  3. Cleavage of His-tag from protein
  4. Bradford assay

Protein complex assembly and activation:
  1. Co-incubation
  2. Activation of nuclease activity
  3. In vitro transcription of target and guide RNA

Product readout:
  1. Universal lateral flow dipsticks for the detection of biotin and FAM-labeled RNA-reporters
  2. Test of RNase presence in spit and urine
  3. RNase degradation in urine and spit
  4. Proteinase inhibition

Protocols

Here you can find all the protocols we have followed in the wet lab during this project. Some of the protocols are for basic laboratory practice as making LB broth and agar as well as agarose gels. These protocols are therefore not mentioned in the experiments section above.


Procedure:
  1. Dissolve 3,5 g of LB-agar per 100 mL of milli-Q.
  2. Autoclave the LB-agar solution.
  3. Cool the agar down to room temperature (or approximate <50°C).
    4. If no antibiotics is needed skip step 4.
  4. Add antibiotic. (e.g. volume of antibiotics needed = (desired volume of antibiotics • total volume) / Concentration of antibiotic).
  5. Pour the LB-agar in empty sterile Petri dishes under sterile conditions (close to a flame).
  6. Let the agar Petri dishes dry before storing them at 4°C.


Procedure:
  1. Suspend 20 g of LB broth in 1 L of distilled water.
  2. Autoclave for 15 minutes at 121 °C.


Procedure:
  1. Fill 5 ml of LB media into an inoculation tube.
  2. Add antibiotic to appropriate concentration.
  3. Take an agar plate with appropriate medium and antibiotic.
  4. Scrape off a single colony from agar plate and mix into the LB-filled inoculation tube.
  5. Vortex medium with colony.
  6. Place inoculation tube in incubator set to 37°C and leave for 16 hours.


Reagents:
  • Agarose powder
  • 1x TAE buffer
  • Ethidium bromide
Procedure (example given for 1% agarose gel):
  1. Add 1g agarose powder per 10 mL TAE buffer to an erlenmeyer flask.
  2. Swirl the flask around, so no large clumps are left. Cover the flask with (PAPER)
  3. Dissolve the agarose in the buffer by heating up in a microwave oven, making sure to take the flask out every 2 minutes, to see if particles are left.
  4. Let the flask cool for 2-3 minutes.
  5. Add 4 g ethidium bromide per 100 mL gel and swirl the flask, to disperse it thrpughout the flask.
  6. Place the flask in a 60 C cabinet until use.


Reagents:
  • Agarose gel with ethidium bromide
  • DNA samples
  • Loading dye
  • DNA ladder
  • (TAE buffer)
Procedure:
  1. Tape up an agarose mold, making sure that no leaks are present.
  2. Add the relevant comb and pour in the agarose gel.
  3. Once the gel has stiffened, carefully take out the comb and remove the tape from the mold.
  4. Transfer to an electroforesis table.


Making CaCl2 competent cells (E. coli)
  1. Streak cells on LB plates and grow at 37°C O/N.
  2. Inoculate 5mL LB media with a single colony and grow at 37°C O/N.
  3. Inoculate 20mL LB media with 20µL O/N-culture (1:1000 dilution).
  4. Grow cells at 37°C (shaking), until OD450= 0.5 (~3 hours).
  5. Transfer cells to 50mL tubes and place cells on ice for >20min.
  6. Spin down cells at 4°C for 10min at 3000g (4830rpm).
    7. Cells must remain cold for the rest of the procedure e.g. transport tubes on ice and resuspend on ice.
  7. Pour off media and resuspend (gently) in 10mL ice-cold 0.1 M MgCl2.
  8. Spin down cells at 4°C for 10min at 3000g (4830rpm).
  9. Pour off media and resuspend cells (gently) in 2mL ice-cold 0.1M CaCl2. Incubate >1hour on ice, but O/N at 4°C is better
  10. Use as they are or add 0.5mL 50% glycerol and transfer 100 µL into Eppendorf tubes placed on dry-ice for long term storage.

Transformation
  1. Thaw CCC on ice (5-10min).
  2. Add DNA* and incubate 10-30min on ice.
  3. Heatshock the cells at 42°C for 90sec. on a heatblock or waterbath
  4. Move the cells back to ice for 2min.
  5. Add 500µL LB (RT)
  6. Incubate at 37°C 60min (preferably horizontal shaking).
  7. Plate 100µL and 500 µL on LB-plates w. antibiotics.
  8. Grow cells o/n at 37°C.
*Max 10 uL; plasmids: 1-10ng and ligation ~25ng


Procedure:
Preparation of E. coli culture:
  1. Add >5mL Lb-media (depends on finale desired volume of transformation) to a bulb
  2. Add specific E. coli strain (possible with a pipette tip) to the bulb
  3. Incubate at 37°C and swirling until abobance for OD600 is between 0,3-0,5

Preparation of TSB buffer:
  1. Add the following components to a 15 mL falcon tube:
    - PEG 3,350 1 g,
    - DMSO 500 µL,
    - MgCl 2 (1M) 200 µL, (OR MgCl 2 6H 2 O 203 g)
    - Fill LB-media up to 10 mL
  2. Mix roughly until reagents are completely dissolved
  3. Transfer the solution to a new falcon tube through a sterile filter with a syringe

TSB transformation:
  1. Spin 1mL E.coli (at OD600: 0,3-0,5) at 3000g at 4°C, remove the supernatant.
  2. Dissolve pellet in 200 μL prepared TSB buffer
  3. Add plasmid (>1000 pg/mL), and incubate at ice bath for 30 min
  4. Transfer from ice to heating block at 42°C for 45 sec, after set heating block to 37°C
  5. Add 1mL fort. LB, and let the sample rest to phenotypical expression at 37°C for 0-2 hrs.
  6. Spin 3000g for 5 min
  7. Remove 90% of supernatant, dissolve the pellet in the rest supernatant
  8. Plate on agar plate with appropriate antibiotic


It is important to consider that the expressing of the gene/protein of interest happens in a bacterium which have high expression of protein. e.g. E.coli ER2566.
Procedure:
  1. Inoculate appropriate amount of LB-medium (depends on desired final yield of protein– for this protocol 200mL is applied) with relevant antibiotic and with O/N. culture.
  2. Grow at 37°C with vigorous shaking until end of exponential phase is reached (OD: 0,6-0,75). (It is possible to collect 1 mL, for later control and comparison)
  3. Induce with IPTG for 3-4 hours (final concentration of IPTG: 0,5-1 mM). (It is possible to collect 1 mL, for later control and comparison).
  4. Harvest the cells by centrifugation at ≈10.000g, 4°C for 10 minutes. Discard the supernatant and resuspend the pellet in 100 mL TMN. Then centrifuge again with same settings as above. Discard the supernatant again and resuspend the pellet in 8 mL TMN-buffer.
  5. Sonicate on ice: 5X 30 sec with pause > 30 sec for each. (settings for sonicator: Hold, constant and 2,5).
  6. Centrifuge the lysates at 8.000g at 4°C for 10 min. Decent the supernatant to a new tube and repeat the centrifugation step. Adjust the supernatant so it gets the same concentration of ions as the wash buffer by adding the following:
    Stock solutions Add Final conc.
    50% glycerol 2,8 mL 10%
    1M NH4Cl 2,7 mL 250 mM
    1M Tris-HCl pH8 0,12 mL 20 mM
    1M mg(O Ac) 0,06 mL 10 mM
    2M Imidazol 0,14 mL 20 mM
    14,3M 2-mercaptoethanol 6,6 µL 6 mM
  7. Save 30 µL of the sample and mix with 10µ SDS-PAGE buffer and freeze until SDS-PAGE analysis.

Preparation of resin:
  1. Wash 1 mL Ni-NTA agarose with 2X 10 mL water and 2X 10 mL wash buffer. Next steps are on 4°C.
  2. Add the liquid and mix gently together with the resin centrifuge 2 min at 2000g then remove buffer.
  3. Mix supernatant with 1 mL washed resin and incubate for 1 hour at 4°C on rotating table.
  4. Apply the resin with supernatant to a Poly-Prep Chromatography column and collect 1 mL flow-through.
  5. Wash the column with 10 mL wash buffer and collect 1 mL flow-through
  6. Wash the column with 10 mL pre-elution buffer and collect 1 mL flow-through
  7. Elute the protein by adding 0,5 mL elution buffer, collect all flow-through. Then add 7,5 mL elution buffer collect 1 mL flow-through.
All the collections of ≈ 1mL can be used in SDS-PAGE.

Table for making the different buffer solutions:
Stock solutions Wash buffer 30 mL Pre- elution buffer 10 mL. Elution buffer 8 mL Final conc.
Water 20,99 mL 6,925 mL 5,06 mL -
1M NH4Cl 1,875 mL 0,625 mL 0,5 mL 250 mM
50% glycerol 6 mL L 2 mL 1,6 mL L 10%
1M MgOAc 0,3 mL 0,1 mL 0,08 mL 20 mM
1M Tris-HCl pH8,0 0,6 mL 0,2 mL 0,16 mL 20 mM
2M Imidazol 0,3 mL 0,15 mL 0,6 mL 20/30/150 mM
14,3M 2-mercaptoethanol 12,6 µL 4,2 µL 3,36 µL 6mM


Kit used: GE healthcare/ Cytiva: illustra™ GFX™ PCR DNA and Gel Band Purification Kit.
Procedure:
  1. Add 10µL capture buffer (type 3) per 10 mg agarose gel in a DNase free tube
  2. Heat the tube up to 60 °C and mix occasional
  3. Add 600µL capture buffer (type 3) to/through assembled GFX-microspin, mix and spin 30s at 16.000g, discard flow through. (possible to load more agarose gel, and repeat step 3)
  4. Add 500µL wash buffer (type 1) and spin 30s at 16.000g, discard tube and transfer GFX-microspin to a new DNase free tube.
  5. Add 10-50µL Elution buffer (type 4 -or 6) let sit for 60s and spin 30s at 16.000g
  6. Discard GFX-microspin
  7. Measure the DNA-conc., 230/260, and 260/280
  8. Store DNA-solution at -20°C


Kit used: GE healthcare/ Cytiva: illustra™ GFX™ PCR DNA and Gel Band Purification Kit.
Procedure:
  1. Add 500 µL capture buffer to max. 100 µL sample
  2. Transfer capture buffer + sample mix into GFX-microspin column and spin 30s at 16.000g. Discard flow-through.
  3. Add 500µL wash buffer (type 1) and spin 30s at 16.000g, discard tube and transfer GFX-microspin column to a new DNase free tube.
  4. Add 10-50µL Elution buffer (type 4 -or 6) let sit for 60s and spin 30s at 16.000g
  5. Discard GFX-microspin
  6. Measure the DNA-conc., 230/260, and 260/280
  7. Store DNA-solution at -20°C


Procedure:
  1. Add reagents to ligation mix
  2. Leave the ligation mix overnight at 16°C
  3. Use ligation solution for transformations
Reagents Volume
10x T4 DNA ligase buffer 2 µL
T4 DNA ligase (add last!) 1 µL
PCR product (cut) of each brick which is to be ligated – or 1 part plasmid and 5 part bricks 5 µL or 10 fmol Plasmid, 0, 10 and 20 fmol PCR product
H2O to reach a total volume of 20µL 
While adding digested brick and digested backbone, optimal ligation happens when molar concentration of backbone and brick is approximately the same. Molar concentration is calculated using:
Equation for optimal ligation
Cng/µl: concentration in ng/µl
Cfmol: concentration in fmol/µl
nbp: number of base pairs in the fragment of DNA


Procedure:
  1. Add reagents in the order indicated in the table below.
    # Reagent Plasmid DNA PCR product Genomic DNA
    1 Sterile water x μl (up to total volume) x μl (up to total volume) x μl (up to total volume)
    2 10x FastDigest or 10x FastDigest Green Buffer 2 μl 3 μl 5 μl
    3 DNA Depends on konc. (~1 μg) Depends on konc. (up to ~0.2 μg) Depends on konc. (~5 μg)
    4 FastDigest enzyme 1 μl 1 μl 5 μl
    5 FastAP (only when digesting backbone) 1 μl
    Total volume 20 μl 30 μl 50 μl
  2. Mix and spin down
  3. Incubate at 37°C in a heat block for at least 1 hour.
  4. The enzyme can sometimes be inactivated by heating, this is optional)
  5. If the 10x FastDigest Green buffer was used, the mixture can be added directly to the gel without loading buffer.
  6. Run the gel and purify the appropriate band protocols for gel electrophoresis and DNA purification from gel. If yield is necessary for next procedure, the gel piece containing the product can be purified using the GE miniprep kit procedure from enzymatic reactions.


Reagents for a 20 µL reaction:
  • MilliQ water to a total volume of 50 µL
  • 10 µL 2x High Fidelity Polymerase master mix with HF buffer 
  • 1 µL 10 mM Forward primer
  • 1 µL 10 mM Reverse primer
  • template (amount depends on concentration)
Procedure:
  1. Pipette the largest volume into the PCR tube, followed by the second largest volume and so on, while keeping all reagents not in use on ice.
  2. If multiple PCR reactions are made, create a mastermix of all the reagents, which all the reactions have in common, and alliquot the different reagents to their specific tubes.
  3. Place into the PCR machine, with following instructions:
    Step Temperature Time (min.)
    Step 1 98°C 3:00
    Step 2 98°C 0:15
    Step 3 55°C-65°C 0:30
    Step 4 72°C 0:30 pr. kilobase template
    Step 5 Repeat step 2-4 x34
    Step 6 72°C 0:15
    Step 7 910°C infinite hold


PCR set up for 1 sample:
Notes: Smaller volume than recommended by Bioline Template: 200 ng or Template: 0.5 μl of 50 μl sterile water boiled with some of the bacteria colony or mix directly into the PCR tube with the microwaved colony
Primers (20 pmol): 0.2 μl each
Amplicon TAQ polymarase (Red), 2x: 5 μl
Water (sterile): 4.1 μl
Total: 10 μl

Procedure:
  1. Add 50 μl sterile water to a 1.5 ml tube – one for each sample.
  2. Dip a pipette tip in a single colony and mix into an Eppendorf tube.
  3. Place the Eppendorf tube in a heating block set at 96 °C for 5 min.
  4. Place the samples on ice.
  5. Mix primers, water and Taq as described under PCR set up. Be sure that all the samples are in the bottom of the PCR tubes by spinning on PCR tube rotator
  6. Place the tubes inside the PCR machine.
  7. Start the appropriate PCR program for Taq or design one yourself.
  8. Keep at 4-5 °C until use, if more than 2 days waiting time place in -20°C

PCR cycling conditions:
Step Temperature Time (min.)
Step 1 98°C 3:00
Step 2 98°C 0:15
Step 3 55°C-65°C 0:30
Step 4 72°C 0:30 pr. kilobase template
Step 5 Repeat step 2-4 x34
Step 6 72°C 0:15
Step 7 910°C infinite hold


Procedure:
  1. Add 250 μL of 50 % glycerol to each sterile freezing tubes.
  2. Vortex ON culture of bacteria.
  3. Add 750 μL ON culture to each of the 3 freezing tubes with glycerol.
  4. Pipette up and down to mix.
  5. Check register sheet for number for the cultures and write on the tubes


Procedure:
  1. After purification, the dilute the Cas proteins and store in protein storage buffer containing (pr 50 mL) with the following:
    • 5 ml Tris HCL (pH 7,5; 0,5 M)
    • 6 ml NaCl (5 M)
    • 2,5 ml glycerol (50%)
    • 100 µl DTT (1 M)
    • 36,4 ml mQ water
    • This was filtered through 0,22 µm 50 ml vacuum filters.
  2. Add an appropriate ratio of Cas and gRNA to a tube and incubate for 10 min at 37°C to ensure complex formation.
  3. Add target RNA or the testing sample into the tubes. They are now ready for detection using either flow strips or RNase Alert.


Procedure:
  1. 100 µl HybriDetect Assay Buffer is added to each reaction tube
  2. 20 µl reaction product is added to the tubes along with 1 µl 10 ng/µl biotin and FAM-labeled RNA-reporter.
  3. The samples are incubated 10 min at 37°C to ensure cleavage of all RNA-reporters before introducing the flow dipsticks.
  4. Dipsticks are added to each reaction tube, incubated for 5 minutes, and then results are interpreted.


Procedure:
  1. 5 µl RNase Alert Lab Test Buffer is added to each sample in one tube of RNase Alert Substrate v2.
  2. 45 µl test solution is added to each tube, vortexed, and mixed.
  3. For the negative control 45 µl nuclease free water is added instead for test solution and for the positive control 40 µl nuclease free water and 5 µl RNase A is added.
  4. The tubes are incubated 30 min to 1 hour at 37°C.
  5. The samples are then looked at under fluorescence light (490/520 nm) or by using a plate fluorometer to see the read-out.


Procedure:
  1. Collect sample fluid.
  2. Cell lysis. Add 10 µL lysis solution to 50 V urine.
  3. Add proteinase K to a concentration of 400 µg/mL.
  4. Vortex and spin down three times.
  5. Incubate at 55°C for 3 hours.
  6. Add PMSF to a concentration of 1 mM.


The protocol from the GenElute Plasmid Miniprep Kit by Sigma-Aldrich was used throughout the project and can be found here.


Procedure:
  1. Turn on the compressor.
  2. Put the samples in the Speedy Vac with open lid.
  3. Close the lid on the Speedy Vac and press run.
  4. Vacuum the Speedy Vac by turning the upper green switch to the mark.
  5. When the Speedy Vac has run for 15 min. or until the samples are concentrated enough, the vacuum is closed by turning the upper green switch back to start position so air again enters the Speedy Vac.
  6. Turn off the Speedy Vac, when it has stopped entering air.
  7. Open the Speedy Vac and take out the samples.
  8. Turn off the compressor.


Procedure:
  1. Make BSA protein standard dilutions of 2.0 µg/mL, 1.5 µg/mL, 1.0 µg/mL, 0.5 µg/mL, 0.25 µg/mL og 0.0 µg/mL (pure milliQ H2O).
  2. Dilute 5x Biorad reagent down to 1x.
  3. Make a 5x and 10x dilution of protein sample by transferring 5 µL to 20 and 45 µL milliQ water, respectively
  4. Prepare an eppendorf tube pr. BSA standard as well as protein sample and add 1 mL 1x biorad reagent to the tubes.
  5. Transfer 5 µL of each BSA standard as well as protein sample to the respective eppendorf tubes and incubate at room temperature in 30 minutes.
  6. Calibrate a spectrophotometer with a sample of 1 mL pure Biorad reagent.
  7. Measure OD595.
  8. Make a standard curve and use this to determine protein concentration.


Procedure:
  1. Add the following to a RNase free tube.
    • Protein of interest contains SUMO-tag 20 µg
    • 10X SUMO Protease Buffer +/- Salt 20 μl
    • Water to 190 μl
    • UMO Protease 10 µL
  2. Incubate ≈ 2 hours at appropriate temperature (Cas12a or Cas13a 30°C).
  3. Confirm SUMO-cleaving by SDS-PAGE utilizing a suitable gel


This protocol has 4 different subparts and can be performed over several days.
Part 1 - Anneal ssDNA oligo's
  1. Make a 2x Annealing Buffer
    • 20 mM Tris, pH 7.5-8.0
    • 100 mM NaCl
    • 2 mM EDTA
  2. Mix the following in a PCR tube:
    • 20 µL ssDNA oligo #1 (e.g., T7-Scaffold-ERG)
    • 20 µL ssDNA oligo #2 (e.g., T7-Scaffold-ERG_REV)
    • 40 µL 2x Annealing Buffer
  3. Put it in the PCR machine with following program:
    1. 95 °C for 2 min
    2. Cool slowly to 25°C over 45 min.
    3. Cool to 4 °C for temporary storage.
  4. Centrifuge PCR tubes.

Part 2 – T7 Transkription with MEGAscript Kit
  1. Mix the following reagents (for small RNAs) in an 1.5 mL Eppendorf tubes (RNase free)
    • 2 µL ATP solution
    • 2 µL CTP solution
    • 2 µL GTP solution
    • 2 µL UTP solution
    • 2 µL 10x Reaction Buffer
    • 250 ng (0.25 µg) Template
    • 2 µL Enzyme mix
    • Nuclease-free Water à 20 µL
  2. Mix Thoroughly
  3. Incubate at 37 °C over-night (around 16 hours )
  4. Add 1 µL TURBO DNase, mix well and incubate 15 min at 37 °C.
  5. Keep on ICE or store at -20 or -80 °C.

Part 3 – Run RNA transcripts on a 8 % polyacrylamide gel
  1. Cast polyacrylamide gel, and let it polymerize for at least 45 min.
  2. Prepare samples by adding RNA loading dye to your samples (5 µL to a 20 µL sample).
  3. When gel is polymerized, prepare the gel electrophoresis system, and pre-run the gel for 20 min at 300 V.
  4. Clean up your wells with a syringe before loading your samples.
  5. Load your samples, and run the gel for 1 h 30 min.
  6. Remove the glass plates and transfer the gel to UV-transparent plastic wrap (cover both sides).

Part 4 – Extract RNA from gel and precipitate
  1. Put the wrapped-in gel onto a TLC plate and use a UV lamp to detect your RNA bands.
  2. Mark your RNA bands, cut out the gel piece and put it into a RNase-free 1.5 mL Eppendorf tube with safe-lock.
  3. Cover the gel pieces with 2 M NH4-Acetate (maximum 500 µL) and incubate over-night at 4 °C with shaking.
  4. Transfer the NH4-Acetate+RNA solution to 3x volume of 96 % EtOH + 25 µL 3 M NaAcetate (use a RNase-free 2 mL Eppendorf tube). Put it on ice for at least 1 hour (or ON in -20 °C freezer).
  5. Spin down a max speed for 45 min, at 4 °C.
  6. Carefully remove supernatant.
  7. Wash your RNA pellet by adding 150 µL 70 % EtOH (cold) and invert the tube a couple of times (do not resuspend RNA pellet – RNA pellet may loosen itself from the bottom of the tube. Be careful not to lose it).
  8. Remove supernatant, and wash again with 150 µL 96 % EtOH (cold) and invert the tube a couple of times (do not resuspend RNA pellet).
  9. Remove supernatant, and air dry the pellets.
  10. Add RNase-free miliQ H2O (30-100 µL).
  11. Measure RNA concentration with Denovix.
  12. Store at -20/-80 °C.