EXPERIMENTS

LYSIS BUFFER X5 STOCK

• 250 mM Tris-HCl
• 500 mM NaCl
• 5% v/v Glycerol
• 1 mM DTT

Stored at 4 ℃ until use.

WASHING BUFFER

• 50 mM Tris-HCl
• 500 mM NaCl
• 5% v/v Glycerol
• 1 mM DTT
• 25 mM Imidazole

Stored at 4 ℃ until use.

ELUTION BUFFER

• 50 mM Tris-HCl
• 500 mM NaCl
• 5% v/v Glycerol
• 250 mM Imidazole

Stored at 4 ℃ until use.

GEL FILTRATION

• 20 mM Tris-HCl
• 200 mM KCl
• 5% v/v Glycerol

Stored at 4 ℃ until use.

REACTION BUFFER

• 10 mM Tris-HCl
• 50 mM KCl
• 1.5 mM MgCl₂

SOC MEDIUM (1 L)

• 20 g tryptone
• 1.25 g yeast extract
• 0.125 g sodium chloride (NaCl)
• 0.046 g potassium chloride (KCl)
• Adjust pH to 7.5 with NaOH
• 1 M sterile-filtered glucose
• 2 M magnesium chloride (MgCl₂)

LB BROTH

Dissolve 25g LB Broth in one liter deionized water and stir until clumps are gone.

Transfer to an Erlenmeyer flask that is at least twice the volume of the liquid.

Autoclave the mixture.

LB – AGAR PLATES

1) For the LB – Agar mixture:

• 1 L distilled water
• 10 g/L NaCl
• 10 g/L Tryptone
• 5 g/L Yeast Extract
• 15 g/L Agar

And mix until everything is dissolved.

2) Autoclave

3) Allow to cool at approximately 55 degrees and add 25mg/mL of Chloramphenicol while swirling

4) Pour 20mL of LB – agar per petri dish while working in flame for sterile conditions

5) Allow the plates to cool for 30-60min until the solidify

6) Seal the plates using parafilm and store in -4 conditions

TRANSFORMATION

1) Thaw the competent cells on ice for 30 minutes.

2) In an Eppendorf tube, gently mix 50 µL of Rosetta 2(DE3)pLysS Competent Cells with 1–5 µg of DNA.

3) Incubate the mixture on ice for 2 minutes.

4) Heat shock the cells at 42 °C for 30 seconds.

5) Immediately place the tube on ice for 5 minutes.

6) Add 950 µL of SOC medium under sterile conditions.

7) Incubate at 30 °C, 250 rpm, for 1 hour.

8) Plate 50–500 µL of the transformed cells onto selective agar plates using a sterile glass pipette.

9) Incubate the plates at 37 °C for 24 hours.

FRAGMENT PCR PROTOCOL

PCR (Polymerase Chain Reaction) is a technique used to amplify a template DNA by cycling through denaturation, annealing, and extension phases with a thermostable DNA polymerase.

Master mix for total volume = 25 µL

Component	Amount / Conc.
Q5 High Fidelity 2X Master Mix	12.5 µL
Forward primer	10 µM
Reverse primer	10 µM
Template DNA	1 ng – 1 µg
Nuclease-free water	Up to 25 µL

PCR cycling temperatures

Step	Temperature (°C)	Time	Number of cycles
Initial denaturation	98	30 s	1
Denaturation	98	10 s	25–40
Annealing	68–72(gradient)	30 s	25–40
Extension	72	30 s/kb → ~1 min 30 s	25–40
Final extension	72	2 min	1
Hold	4	∞	1

AGAROSE GEL PREPARATION AND ELECTOPHORESIS

Agarose gel electrophoresis is used to separate DNA fragments based on their size. The percentage of agarose gel is chosen according to the length of the DNA fragments to achieve optimal resolution.

Linear DNA (bp)	% Gel
1,000 – 30,000 bp	0.5%
800 – 12,000 bp	0.7%
500 – 10,000 bp	1.0%
400 – 7,000 bp	1.2%
200 – 3,000 bp	1.5%

Making the agarose gel

Gel %	Agarose	TAE buffer
0.7%	0.7 g	100 mL
1.5%	1.5 g	100 mL
3%	3 g	100 mL

1) Mix the desired amount of agarose in 100 mL of TAE buffer.

2) Microwave for 1–3 minutes until fully dissolved.

3) Allow the solution to cool to a temperature safe to handle (can also cool under running tap water).

4) Add 1 μL of Gel Red per 100 mL of agarose solution and mix gently.

5) Pour the solution into a gel casting tray with the appropriate comb and let it set for 20–30 minutes.

Running the gel

1) Add 5 uL of 100 bp DNA ladder to the first well.

2) Add 3 uL of 6x Orange G loading dye into all the wells and then add 5 uL of the DNA sample to each well (in our case we had 6 samples – 3 of each fragment).

3) Run at 100 V for 30–45 minutes.

Visualizing the gel

View the gel under a UV light or a blue light transilluminator

GEL EXTRACTION FROM AGAROSE GEL

For gel extraction from our gel we used the NucleoSpin® Gel and PCR Clean- up kit from Macherey – Nagel.

NucleoSpin Gel and PCR Clean-up — Instructions (PDF)

VECTOR OVERHANGS PCR

For this PCR, we used specific primers designed to linearize the DNA and generate the desired overhangs. This procedure prepares our vector for Gibson assembly.

Master mix

Component	Total volume = 50 µL	Master mix for ×4 samples
Q5 Master Mix	25 µL	100 µL
10 µM forward primer	2.5 µL	10 µL
10 µM reverse primer	2.5 µL	10 µL
DNA template	5 µL	20 µL
DMSO	3%	3%
Nuclease-free water	Up to 50 µL	Up to 200 µL

PCR cycling temperatures

Step	Temperature (°C)	Time	Number of cycles
Initial denaturation	98	30 s	1
Denaturation	98	10 s	25–40
Annealing	68–72(gradient)	30 s	25–40
Extension	72	2 min 30 s	25–40
Final extension	72	5 min	1
Hold	4	∞	1

GIBSON ASSEMBLY

Gibson Assembly is a molecular cloning method used to join multiple DNA fragments that have overlapping ends. Unlike traditional restriction enzyme-based cloning, it does not require specific restriction sites for fragment assembly.

Master mix for 1 fragment insertion

Component	Amount
DNA	0.03–0.2 pmol
Gibson Assembly Master Mix (2×)	10 µL
Nuclease-free water	Up to 20 µL

Incubate the reaction at 50 °C for 15–60 minutes.

DIGESTION

Restriction endonucleases are commonly used to clone DNA fragments into a plasmid backbone.

For V_total = 20 µL

Component	Amount
EcoRV	0.5 µL
SmartCut Buffer	2 µL
DNA	5 µL
Nuclease-free water	12.5 µL

Incubate the mixture at 37 °C for 1 hour.

NATIVE-PAGE 10%

Gel preparation

• 2.5mL 38:2 Acrylamide:Bis Acrylamide
• 5mL of 1.5M Tris-HCl (pH=8.8)
• 7.5ml TBE 1x
• 40μL 10% w/v APS
• 10μL TEMED

Procedure

1) Mix all components except TEMED by vortexing.

2) Add TEMED and vortex.

3) Pour on top of polymerized separating gel.

4) Insert comb carefully to form wells.

5) Allow polymerization 20–30 min at room temperature.

6) Run the Gel with TBE buffer 1x, float constant voltage 5 V/cm (50 V) for 5 min to allow the DNA to enter gel, then increase the voltage to 10 V/cm (100 V) for the duration of the run.

PROTEIN EXPRESSION

• Plasmid containing the CRISPR/Cas13a protein
• Rosetta 2(DE3)pLysS Competent Cells
• Chloramphenicol for selection

Procedure

Plasmid Transformation & Overnight Culture:

1. Transform the plasmid into Rosetta 2(DE3)pLysS Competent Cells.

2. Plate on chloramphenicol-containing agar and incubate overnight at 37°C.

3. Pick a single colony and inoculate 5 mL LB + chloramphenicol; incubate overnight at 37°C with shaking.

4. Transfer the overnight culture into 500 mL LB broth; incubate at 37°C with shaking.

Protein Expression:

• Using the constitutive promoter BBa_J23100, protein expression occurs during cell growth.
• Harvest cells when OD₆₀₀ ≈ 3.

Protein Harvesting:

• Centrifuge at 14,000 rpm for 15 min to collect the cell pellet.
• Store at –80°C with glycerol.

HIS TAG PROTEIN PURIFICATION

For the protein purification we used Ni-NTA

1. Cell Lysis:

• Resuspend the harvested cell pellet in lysis buffer
• Lyse cells via sonication and centrifuge at ~14,000 × g for 20–30 min at 4°C. Collect the clarified lysate.

2. Column Preparation:

• Equilibrate the Ni-NTA column with 5–10 column volumes of binding buffer.

3. Protein Binding:

• Load the clarified lysate onto the column. His-tagged protein binds the resin.

4. Wash:

• Wash the column with 5–10 column volumes of wash buffer to remove non-specifically bound proteins.

5. Elution:

• Elute the protein with elution buffer containing 250–500 mM imidazole.

6. Storage:

• –80°C with glycerol for long-term.

CRISPR/Cas13a REACTION

All the hairpin probes were formed by heating the sample to 95°C for 5 min, and then slowly cooling to 25°C

(total volume = 20 μL):

• 1 μL Cas13a protein (0.4 μM)
• 1 μL crRNA (0.4 μM)
• 4 μL H0 hairpin (5 μM)
• 2 μL of miRNA at the desired concentration
• Add reaction buffer to bring the final volume to 20 μL

Incubate the reaction at 37°C for 1 hour.

CHA REACTION

Transfer the reactants from each reaction tube to new tubes. Heat the hairpin probes to 95°C for 5 minutes, then allow them to slowly cool to 25°C to ensure proper formation.

(total volume = 100μL)

• 5 µL hairpin 1 (1 µM)
• 5 µL hairpin 2 (1 µM)
• 20 µL CRISPR/Cas13a reaction
• 70 µL PBS (10×)

The Catalytic Hairpin Assembly took place at 37 degrees for 3 hours.

MICRORNA ISOLATION FROM SERUM

MicroRNAs were isolated using the miRNeasy Serum/Plasma Advanced Kit (Qiagen), following the manufacturer's protocol "Purification of Total RNA, Including miRNA, From Serum and Plasma."

miRNeasy Serum/Plasma Advanced Kit (Qiagen) (PDF)

REVERSE TRANSCRIPTION PCR (RT-PCR)

Reverse transcription PCR (RT-PCR) combines the conversion of RNA into complementary DNA (cDNA) with the amplification of specific DNA targets by PCR. In this case the method was used to quantify microRNAs. For cDNA synthesis, we used the first-strand cDNA synthesis protocol provided in Qiagen's miRCURY® LNA® miRNA SYBR® Green PCR kit, along with specific miRNA primers from the same manufacturer.

miRCURY® LNA® miRNA SYBR® Green PCR kit (Qiagen) (PDF)

REAL-TIME PCR (qPCR)

Quantitative PCR was performed using the miRCURY® LNA® miRNA SYBR® Green PCR kit (Qiagen), following the manufacturer's protocol "Quantitative, Real-Time PCR Using Individual miRCURY LNA miRNA PCR Assays."

miRCURY® LNA® miRNA SYBR® Green PCR kit (Qiagen) (PDF)