Team:Yonsei Korea/Experiments

IGEM_YONSEI

In the Laboratory:

The Science
Behind It All

EXPERIMENTS
DNAzyme CLEAVAGE ASSAY        
PART 1: REACTION BUFFER PREPARATION
  1. A. Materials
    1. : 1M HEPES (pH 7.4), KCl, NaCl, MgCl2⋅6H2O, MnCl2⋅4H2O, CuCl2⋅2H2O, Milli-Q water, 15mL tube, vortex, weighing paper, plastic spoons, scale, labeling tape
  2. B. Procedure
    1. : Prepare 10000 µL of 5x reaction buffer based on the calculations below.
      1. - Since the mass of CuCl2 2H2O to make 0.25mM CuCl2 2H2O for 5x reaction buffer is too small, make a separate 100mM CuCl2 2H2O solution first. Use this as a stock solution to prepare 0.25mM CuCl2 2H2O for the 5x reaction buffer.
      2. - When preparing the solutions, make sure to label each solution.
      3. - Vortex the solution until the solids are all dissolved.
Volume
needed (μL)
Stock (M) Mass
needed (g)
Molar mass
(g/gmol)
Solution 1: 5x Reaction Buffer
250mM HEPES (pH 7.4) 2500 1
2M NaCl 1.169 58.44
0.5M KCl 0.373 74.55
50mM MgCl2 6H2O 0.102 203.3
37.5mM MnCl2 4H2O 0.074 197.7
0.25mM CuCl2 2H2O 25 0.1
Buffer total volume 10000 (= 10mL)
 
Solution 2: 100mM CuCl2 2H2O solution
CuCl2 2H2O 0.102 170.48
Total volume 6000 (= 6mL)
PART 2: DNAzyme CLEAVAGE REACTION
  1. A. Materials
    1. : DNAzymeA, GeneA, buffer, Milli-Q water, DEPC H2O, test tube, vortex, 1.5mL microcentrifuge tube, 1M HEPES (pH 7.4), pipettes, pipette tips, heat block
  2. B. Procedure
    1. **Note: keep the samples in ice.
      1. 1. Preparation of 10 µM DNAzyme A: Dilute your stock sample depending on the stock concentration. Use DEPC H2O in dilution.
      2. 2. Preparation of 1µM Substrate (Gene A): Dilute your stock sample depending on the stock concentration. Use DEPC H2O in dilution.
      3. 3. Dilute 5x reaction buffer (prepared from Part 1 above) to make 2x reaction buffer. Use Milli Q water to dilute the reaction buffer.
      4. 4. In a single 1.5mL microcentrifuge tube, insert the following:
        Ingredient Volume (µL)
        10 µM DNAzyme 2
        1 µM Substrate 2
        2x reaction buffer 2
        Milli-Q water 14
        Total volume 20
      5. 5. Heat the DNAzyme-substrate mixture at 90°C for 5 minutes using a water bath.
      6. 6. Cool the mixture at 25°C room temperature for 10-15 minutes.
      7. 7. Incubate at 37℃ for 16 hours (overnight).
PART 3: STOPPING THE CLEAVAGE REACTION WITH EDTAN
  1. A. Materials
    1. EDTA, 20µl pipette, 20µl pipette tips
  2. B. Procedure
    1. 1. Take out the samples from the 37°C incubator after 16 hours.
    2. 2. Using a 20µl pipette, take 2µl of EDTA and insert it into all of the samples to stop the DNAzyme cleavage activity.
    3. 3. Store the samples and solutions in the fridge.
AGAROSE GEL ELECTROPHORESIS        
  1. A. Materials
    1. Agarose powder, 0.5x TAE buffer, comb, gel tray, 6x loading dye, 1kb DNA ladder plastic plate, scale, spoon, glass bottle, plastic protective holders, graduated cylinder, 20µl pipette, 20µl pipette tips, ice bucket, vortex mixer, spin down machine, power supply (for applying voltage to the gel electrophoresis), rack, labeling tape, microwave, gel box, UV light lamp.
  2. B. Procedure
    1. 1. Take the gel tray and the comb, and assemble the gel cassette.
      **Note: choose the comb that has the desired number of wells, depending on the number of samples.
    2. 2. Take the dried plastic plate and zero the scale.
    3. 3. Making 1% agarose gel:
      1. a. Measure 0.4g of agarose powder with the plastic spoon.
      2. b. Put the measured agarose powder into the glass bottle.
      3. c. Measure 40mL of 0.5x TAE buffer using the 100mL graduated cylinder.
      4. d. Pour the TAE buffer into the glass bottle, which has the agarose powder inside.
        **Note: don’t close the glass bottle lid tightly, since it might burst during the microwave.
    4. 4. Microwave the mixture for 2 minutes and 30 seconds.
    5. 5. Use the protective glove to take out the heated glass bottle out of the microwave.
    6. 6. Cool the agarose mixture on the bench until it is possible to touch it with your bare hands for 3 seconds.
    7. 7. Pour the cooled agarose mixture into the tray that has been set up in step #1.
    8. 8. Leave it for 30 minutes, so that the gel will solidify.
    9. 9. While the gel is being solidified, prepare your loading samples:
      1. a. Take the 6x loading dye out of the refrigerator and place it in the ice.
      2. b. Take 4.5µl of 6x loading dye and put it into each sample (the volume of each sample is 22µl).
        **Note 1: after inserting the 6x loading dye into our samples, we have to change the pipette tip.
        **Note 2: the volume of the 6x loading dye will depend on the sample volume.
      3. c. Vortex and spin down the mixture until it is fully mixed.
        **Note: before loading the mixture into the wells, vortex and spin down once more.
      4. d. After using the 6x loading dye, make sure to put it back into the refrigerator as soon as possible, since it is sensitive to temperature and light.
    10. 10. Once the gel is solidified, take out the comb. Take out the gel and place it inside the gel box. Make sure the gel is fully covered with the 0.5x TAE buffer.
    11. 11. Load 3ul of the 1kb DNA ladder inside the 1st lane. Leave the 2nd lane empty.
    12. 12. Load the prepared samples into the wells, starting from the 3rd lane.
      **Note: make sure to keep the order of the samples, so that we are not confused.
    13. 13. Close the lid and connect the power supply.
    14. 14. Set the power supply to 30 minutes and 135V.
    15. 15. Visualize the results, using the UV light lamp:
      1. a. When the gel electrophoresis is complete, turn off the power.
      2. b. Use the transparent, plastic tray to carry your gel.
      3. c. Turn on the UV light visualization machine.
      4. d. Set the machine to “Nucleic Acid Gel”
    16. 16. After viewing the results, clean up the machine:
      1. a. After taking out the gel, clean the machine with 4% EtOH and Kimtech wipers (tissue).
      2. b. Turn off the machine.
PAGE        
  1. A. Equipment
    1. Glass plates (small and tall plates) with integrated spacers, casting frame, casting stand, plastic combs, electrophoresis chamber, power supply, lid, serological pipette, an automatic pipette aid.
  2. A. Reagents
    1. 40% acrylamide gel, 10% APS (Ammonium Persulfate), TEMED, 5x TBE (Tris Boric EDTA) buffer, MilliQ water, samples, 10 bp DNA ladder.
  3. A. Procedure
    1. 1. Assembling the gel casting apparatus:
      1. 1.1. Take the short and tall glass plates and make sure they are dry. If not, use a lint free wipes to dry them.
      2. 1.2. Take the casting frame and place it on the bench with its clamps open (perpendicular to the frame) and facing you.
      3. 1.3. Insert the glass plates into the frame. Place the taller glass plate with its spacer and UP arrows up and the short glass plate in front of the casting frame. Make sure that the botted edges of the two plates MUST be even!
      4. 1.4. Secure the glass plates in the casting frame by pushing the two gates of the frame out to the sides.
      5. 1.5. Place the casting frame with glass plates into the casting stand and clamp them.
      6. 1.6. Check to see if the apparatus is fully assembled by pipetting a small amount of D.W. If the glass plates do not leak, you are ready to prepare the gel. Pour the D.W. out by holding the entire casting platform over a sink. Use the lint free tissues to absorb any residual D.W.
    2. 2. Preparing the resolving gel:
      1. 2.1. Take 2 new 15 mL tubes and label them as 15% and 10%.
      2. 2.2. Add all the required reagents according to the given information on the table:
        10% Native PAGE Total volume - 8 mL
        40% acrylamide gel 2.0 mL
        10% APS 80 uL
        TEMED 8 uL
        Milli Q water 5.2 mL
        5x TAE 0.8 mL
        15% Native PAGE Total volume - 8 mL
        40% acrylamide gel 2.3 mL
        10% APS 80 uL
        TEMED 8 uL
        Milli Q water 4.8 mL
        5x TAE 0.8 mL
      3. 2.3. Immediately mix the reagents by vortexing.
      4. 2.4. Pour the gel immediately using the serological pipette and an automatic pipette aid between the two glass plates.
      5. 2.5. Immediately insert the plastic comb (15 wells) into the gel.
      6. 2.6. Leave the gel for 15 min at room temperature to completely harden.
    3. 3. Running the gel:
      1. 3.1. Once the gel is hardened, place it inside the electrophoresis chamber.
      2. 3.2. Make 0.5x TBE buffer by mixing 950 mL of Milli Q water and 50 mL of 10x TBE buffer.
      3. 3.3. Add 0.5x TBE buffer to completely fill the inner chamber between the glass plates.
      4. 3.4. Add 0.5x TBE buffer to the chamber until it matches the 2 gel sign written on the chamber wall.
      5. 3.5. Carefully remove the comb and load the samples into the wells of the gel.
      6. 3.6. Close the lid and connect the cables to the power supply. Make sure to connect the black and red cables to the corresponding colours on the supply.
      7. 3.7. Run the gel at 90V for 2 hours.
    4. 4. Visualising the results:
      1. 4.1. Once the running process is complete, turn off the power and remove the lid carefully.
      2. 4.2. Prepare the tray and pour a small amount of D.W in it.
      3. 4.3. Take the gel cassette carefully and discard the buffer inside the inner chamber.
      4. 4.4. Release the clamps and take the glass plates out.
      5. 4.5. Place the glass plates onto the tray and carefully remove the gel.
      6. 4.6. Visualise the results using the UV machine.
DENATURING PAGE (UREA PAGE)        
  1. A. Equipment
    1. Glass plates (small and tall plates) with integrated spacers, casting frame, casting stand, plastic combs, electrophoresis chamber, power supply, lid, serological pipette, an automatic pipette aid.
  2. A. Reagents
    1. 10% APS (Ammonium Persulfate), TEMED, 5x TBE buffer, formamide, samples.
  3. A. Procedure
    1. 1. Assembling the gel casting apparatus:
      1. 1.1. Take the short and tall glass plates and make sure they are dry. If not, use a lint free wipes to dry them.
      2. 1.2. Take the casting frame and place it on the bench with its clamps open (perpendicular to the frame) and facing you.
      3. 1.3. Insert the glass plates into the frame. Place the taller glass plate with its spacer and UP arrows up and the short glass plate in front of the casting frame. Make sure that the botted edges of the two plates MUST be even!
      4. 1.4. Secure the glass plates in the casting frame by pushing the two gates of the frame out to the sides.
      5. 1.5. Place the casting frame with glass plates into the casting stand and clamp them.
      6. 1.6. Check to see if the apparatus is fully assembled by pipetting a small amount of D.W. If the glass plates do not leak, you are ready to prepare the gel. Pour the D.W. out by holding the entire casting platform over a sink. Use the lint free tissues to absorb any residual D.W.
    2. 2. Preparing the gel:
      1. 2.1. Take a new 15 mL tube.
      2. 2.2. Add all the required reagents according to the given information on the table:
        8% Urea PAGE gel Total volume - 10 mL
        40% acrylamide gel 3.2 mL
        Urea 5.8 mL
        5x TBE buffer 1.2 mL
        10% APS 100 uL
        TEMED 10 uL
      3. 2.3. Immediately mix the reagents by vortexing.
      4. 2.4. Pour the gel immediately using the serological pipette and an automatic pipette aid between the two glass plates.
      5. 2.5. Immediately insert the plastic comb (15 wells) into the gel.
      6. 2.6. Leave the gel for 15 min at room temperature to completely harden.
    3. 3. Preparing the resolving gel:
      1. 3.1. Take the samples out of the storage at -20°C and melt them on ice.
      2. 3.2. Take the formamide and keep it on ice.
      3. 3.3. Set the heat block to heating mode at 90°C.
      4. 3.4. Take new 1,5 mL microcentrifuge tubes and label them according to the experiment condition.
      5. 3.5. Add 1:1 ratio of the samples and formamide to the labeled tubes. For instance, you can add 10 uL of the sample and 10 uL of formamide.
      6. 3.6. Gently vortex the mixture and place it on a heat block at 90°C for 20 minutes to denature dsDNAs.
      7. 3.7. After 20 minutes, take the samples. Cool the samples by using the bench mini centrifuge to spin for 1 minute.
    4. 4. Running the gel:
      1. 4.1. Once the gel is hardened, place it inside the electrophoresis chamber.
      2. 4.2. Make 0.5x TBE buffer by mixing 950 mL of Milli Q water and 50 mL of 10x TBE buffer.
      3. 4.3. Add 0.5x TBE buffer to completely fill the inner chamber between the glass plates.
      4. 4.4. Add 0.5x TBE buffer to the chamber until it matches the 2 gel sign written on the chamber wall.
      5. 4.5. Carefully remove the comb and load the samples into the wells of the gel.
      6. 4.6. Close the lid and connect the cables to the power supply. Make sure to connect the black and red cables to the corresponding colours on the supply.
      7. 4.7. Run the gel at 200V for 40 minutes.
    5. 5. Visualising the results:
      1. 5.1. Once the running process is complete, turn off the power and remove the lid carefully.
      2. 5.2. Prepare the tray and pour a small amount of D.W in it.
      3. 5.3. Take the gel cassette carefully and discard the buffer inside the inner chamber.
      4. 5.4. Release the clamps and take the glass plates out.
      5. 5.5. Place the glass plates onto the tray and carefully remove the gel.
      6. 5.6. Visualise the results using the UV machine.
DNA FUNCTIONALIZED GOLD NANOPARTICLES
(SALT-AGING METHOD)        
  1. A. Equipment
    1. Vortex machine, orbital shaker, oven
  2. A. Materials
    1. Thiolated Sequence 1 (TS1): 3’-tgc cgg tca cgg ccg ctg tc-5’ and thiolated Sequence 2 (TS2): 3’-atc gtt ggg gtg acc gag cc-5’, PBS (pH 8), Na2HPO4, NaH2PO4, NaCl, 1.5mL microcentrifuge tubes, 15mL tube, nap5 columns, labelling tape, metal spatula, weighing paper, purified water

      Synthetic sequences and thiolated sequences (1&2) were purchased from IDT Korea. Gold nanoparticles (30 nm) were purchased from BBI Solutions.
  3. A. Procedure (adapted from reference 1)
    1. 1. Lyophilize 5 nmol thiolated oligonucleotide probe (TS1: 3’-tgc cgg tca cgg ccg ctg tc-5’ and TS2: 3’-atc gtt ggg gtg acc gag cc-5’).
      1. a. Steps 1-20 were conducted for TS1 first and subsequently repeated for TS2.
    2. 2. Prepare 1 ml of 0.1 M DTT solution in the disulfide cleavage buffer.
      1. a. This solution must be made fresh every time.
    3. 3. Add 100 µl DTT solution (as prepared in Step 2) to the 5 nmol of lyophilized DNA, wrap in foil and let stand at room temperature for 2–3 h. Vortex occasionally.
    4. 4. 15 min before the completion of disulfide cleavage, begin flushing a Nap-5 column with NANOpure water. At least three column volumes of NANOpure water must flush through before adding DNA.
    5. 5. Add the 100 µl of DNA to the column after all the water has run through.
    6. 6. Once the 100 µl of DNA has flowed into the column, add 400 µl of NANOpure water to the column and allow it to flow through uncollected.
    7. 7. Then add 950 µl NANOpure water to the column, and collect the flowthrough 3–4 drops at a time in 1.5-ml microcentrifuge tubes.
    8. 8. Use a UV-visible spectrophotometer and the absorbance at 260 nm to determine the DNA location and concentration using Beer’s Law: A = εCι, where ε is molar absorptivity, C is concentration and ι is cell path length.
    9. 9. Rinse a 20-ml EPA vial with ethanol and then NANOpure water; blow dry.
    10. 10. Add 1 ml 13-nm Au-NPs synthesized previously.
    11. 11. Calculate the number of moles of oligonucleotide per tube, and add 4 nmol of the freshly reduced thiolated oligonucleotide to the Au-NPs. Record the volume.
    12. 12. Wrap in foil and place on an orbital shaker overnight at room temperature.
      1. a. PAUSE POINT: This step can be left overnight.
    13. 13. Add phosphate adjustment buffer to the NP solution to obtain a final phosphate concentration of 9 mM.
      1. a. Calculation: 1,000 µl AuNPs + x µl DNA = total volume in µl. (Total volume 1 in µl)/10 = y µl phosphate adjustment buffer needed.
    14. 14. Add surfactant solution to obtain a final SDS concentration of ~0.1% (wt/vol). This helps to keep the particles from aggregating and adds to the efficiency of washing them in future steps.
      1. a. Calculation: 1000 µl AuNPs + x µl DNA + y µl phosphate adjustment buffer = total volume 2. SDS to add = (total volume 2 × 0.1)/10.
    15. 15. Rewrap in foil and place on an orbital shaker for 30 min.
    16. 16. Calculate the volume of salting buffer needed to obtain a final concentration of 0.3 M NaCl.
      1. a. Calculation: (Total volume 2 × 0.3 M)/(2M) = volume of salting buffer needed in microliters. The number of additions of the salting buffer is equal to 6, therefore the amount per addition is equal to the volume of the salting buffer divided by 6.
    17. 17. Over the course of 2 d, make six additions of one-sixth of the total salting buffer needed to reach a final concentration of 0.3 M NaCl. Do the additions while shaking gently or at a low vortex speed.
    18. 18. After the last salt addition, allow the particles to equilibrate overnight. Well-functionalized Au-NPs should be the same color as the un-modified Au-NPs with no visible aggregates.
      1. a. PAUSE POINT: Particles can be stored at room temperature for as long as 1 month in this state.
TARGET DNA/FUNCTIONALIZED GOLD
NANOPARTICLES HYBRIDIZATION        
  1. A. Equipment
    1. Vortex machine, orbital shaker
  2. A. Materials
    1. Synthetic sequence (SS): 5’-acggccagtgccggcgacagctctagcaaccccactggctcgg-3’, PBS (pH 8), 1.5mL microcentrifuge tubes, labelling tape, purified water

      Synthetic sequences and thiolated sequences (1&2) were purchased from IDT Korea.
  3. A. Procedure
    1. 1. Mix in 1.5-ml microcentrifuge tubes: (i) 30 µl PBS (8) buffer; (ii) 10 µl GNPs solution; (iii) 10 µl target solution.
    2. 2. Shake the reactions at room temperature for 45 min. Shake sufficiently so that the particles do not settle.
    3. 3. Use a UV-Visible spectrometer to determine shifts in absorbance of samples.
REFERENCES