Team:HK HCY LFC/Experiments

Team:HK HCY LFC - 20118.igem.org

Experiments to test the DNA nanostructure

Overview:

1. Preparation of the DNA working solutions

2. Testing different combination and the assembly of DNA nanostructure

3. Polyacrylamide Gel Electrophoresis (PAGE)

4. Peroxidase Activity Assay

1. Preparation of the DNA working solutions

Materials:

· DNA

· 10x PBS

· ddH₂O

Procedures:

1. Wipe the bench and your gloves with 70% ethanol.

2. Briefly vortex and centrifuge the oligo stock solutions before dilution.

3. Prepare the followings.

4. Mix well by tapping and briefly centrifuge the tubes.

5. Store at -20^oC.

2. Testing interactions between each DNA strands

Materials:

· diluted DNA

· 1x PBS

Procedures:

1. Tap and centrifuge the prepared working DNA solutions.

2. Prepare the following PCR tubes.

Interactions between two DNA strands

3. Tap and centrifuge the PCR tubes.

4. Incubate the PCR tubes at 95^oC for 5 min and cool down to 25^oC with 0.5^oC drop every 30 seconds using a thermal cycler.

5. Store the solution at -4^oC.

3. Polyacrylamide Gel Electrophoresis (PAGE)

Materials:

· DNA (taken from the thermocycler)

· target RNA

· 1x PBS

· ddH₂O

· loading dye

· 10 bp DNA ladder

· sybr safe DNA stain

For making the 12% polyacrylamide gel:

· 30 % Acrylamide (3900 µL))

· 5x PBS (1560 µL)

· 5x TBE (1560 µL)

· Water (780 µL)

· 10% APS (130 µL)

· TEMED (6.5 µL)

(a) Strand Displacement Reaction for PAGE

1. Prepare the following mixture:

2. Place the tubes in a shaker and incubate them at room temperature for 30 minutes for strand displacement reaction.

(b) Preparation of Solutions for PAGE

1. Dilute all 1μM DNA solutions to be loaded as follows:

1. Dilute all 5μM DNA solutions to be loaded as follows:

3. Dilute the DNA ladder as follows: 1μL DNA ladder (10 bp) + 11μL 1X buffer.

Running PAGE

1. Put the gel in the electrophoresis tank, add 1X TBE buffer and remove the comb.

2. Load 12μL of each of the DNA samples.

3. Run the gel at a constant voltage of 100V for around 60 minutes or until the bands of the dye reach 3/4 of the length of the gel.

4. Use SYBR Safe to post-stain the gel for 15-20 minutes on a shaker and observe the gel under UV light.

4. Peroxidase Activity Assay

Materials:

Prepare the folowing working concentrations of reagenets:

Procedures:

1. Prepare the reaction mixture as follows, following the subsequent steps:

2. Add buffer, DNA nanostructure, target miRNA and hemin and briefly vortex the solution.

3. Incubate at room temperature on shaker for 20-30 minutes, using aluminum foil to cover the tube holder (as hemin is light-sensitive).

4. Add 10μL ABTS and 10μL H₂O₂ and briefly vortex the solution.

5. Transfer the reaction mixtures to a 96-well plate.

6. Make sure that you do not introduce bubbles.

7. Measure the absorbance at 415 nm by a spectrophotometer at 30-second intervals for 20 minutes.

8. Repeat the experiments for 180 nM, 125 nM, 150 nM, 100 nM, 75nM, 50 nM. 15 nM, 10 nM, 0nM of target miRNA.