Experiments to test the DNA nanostructure
· 10x PBS
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 -20oC.
· diluted DNA
· 1x PBS
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 95oC for 5 min and cool down to 25oC with 0.5oC drop every 30 seconds using a thermal cycler.
5. Store the solution at -4oC.
· DNA (taken from the thermocycler)
· target RNA
· 1x PBS
· loading dye
· 10 bp DNA ladder
· sybr safe DNA stain
· 30 % Acrylamide (3900 µL))
· 5x PBS (1560 µL)
· 5x TBE (1560 µL)
· Water (780 µL)
· 10% APS (130 µL)
· TEMED (6.5 µL)
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.
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.
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.
Prepare the folowing working concentrations of reagenets:
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.