Experiments
To demonstrate our calculations simulated by computer, we carried out following experiments. We will show you these experiments in chronological order and let you experience our trial and error and improvements in experimental method.
All the experiments can be categorized into six parts:
a. Acquire single chain DNA sequences used in wet experiments (we got these sequences by our Software Tool).
b. Synthesize DNA in part 1 (in fact we bought them from Sangon Biotech).
c. Dissolve, dilute, mix.
d. Denature and anneal (acquire the results of fluorescence intensity by qRT-PCR simultaneously).
e. Purify and quantify the reactant complexes.
f. Characterization via PAGE
Basic experiment details are as follows:
1. Dissolve, dilute, mix
All the single chain DNAs were dissolved in DEPC-treated water to form 100 M solutions respectively and saved at 4 ℃.
Reactant complexes were annealed together at 20 uM in Tris-acetate-EDTA buffer containing 12.5 mM Mg2+(1×TAE/Mg2+) and saved at 4 ℃ for further experiments.
2. Gel electrophoresis
To examine the products of every kinetics experiments and purify the reactant complexes, 12% non-denaturing PAGE was run at 40 V for about 3.5 hours. After staining with GelRed (Biotium, 1:10000) for 30 min, the gel was scanned on a gel imaging system (Tanon 3500R).
Formulation for 12% native PAGE is as follows:
| 29:1 30% Acrylamide/bis |
Deionized Water |
5xTBE Buffer |
10% APS |
TEMED |
Total Volume |
| 4mL |
3.93mL |
2mL |
0.07mL |
0.007mL |
10mL |
3. Reactant complexes purification and quantification
After acquiring the reactant complexes, we used them to carry out the polyacrylamide gel electrophoresis and obtained the target stripes cut down from the gel with the assistance of a gel imaging system (Tanon 3500R).
4. Kinetics experiments
Denaturing and annealing were performed in a quantitative real-time PCR (qRT-PCR) machine, first heating up to 95 oC, then slowly cooling down to 25 ℃ at the rate of 1 ℃/min, and finally holding at 20 ℃ for 2－4 hours.
For the same sample, we tried two concentrations of reactants, that is, 1 uM and 0.1 uM. For each concentration, we used three reaction cups to carry out the experiments, 20 uL in each cup.
Reactant complexes were synthesized with the reactant amount at 20 uM.
Experiment process are as follows:
1. Synthesis of reactant complexes
Reactant complexes were annealed together at 20 uM in Tris-acetate-EDTA buffer containing 12.5 mM Mg2+(1×TAE/Mg2+) and saved at 4 ℃ for further experiments. Volumes for all the reaction system here at one time are all 50 uL. The concrete details are shown in table (XX).
| 1Gi (Reactant Complex for Addition) |
| 1Gia (100 uM) |
10uL |
| 1Gib (100 uM) |
10uL |
| Deionized Water |
25uL |
| 10×TAE Buffer |
5uL |
| 1Ti (Reactant Complex for Addition) |
| 1Tia (100 uM) |
10uL |
| 1Tib (100 uM) |
10uL |
| 1Tic (100 uM) |
10uL |
| Deionized Water |
15uL |
| 10×TAE Buffer |
5uL |
| 2Li (Reactant Complex for Addition) |
| 2Lia1 (100 uM) |
10uL |
| 2Lia2 (100 uM) |
10uL |
| 2Lib (100 uM) |
10uL |
| Deionized Water |
15uL |
| 10×TAE Buffer |
5uL |
| 2Ti (Reactant Complex for Addition) |
| 1Tia (100 uM) |
10uL |
| 1Tib (100 uM) |
10uL |
| Deionized Water |
25uL |
| 10×TAE Buffer |
5uL |
| 3Li (Reactant Complex for Addition) |
| 3Lia1 (100 uM) |
10uL |
| 3Lia2 (100 uM) |
10uL |
| 3Lib (100 uM) |
10uL |
| Deionized Water |
15uL |
| 10×TAE Buffer |
5uL |
| 3Li (Reactant Complex for Addition) |
| 3Lia1 (100 uM) |
10uL |
| 3Lia2 (100 uM) |
10uL |
| 3Lib (100 uM) |
10uL |
| Deionized Water |
15uL |
| 10×TAE Buffer |
5uL |
| 3Ti (Reactant Complex for Addition) |
| 3Tic (100 uM) |
10uL |
| 3Tid (100 uM) |
10uL |
| 3a (100 uM) |
10uL |
| 3b |
10uL |
| Deionized Water |
5uL |
| 10×TAE Buffer |
5uL |
| 3Wi (Reactant Complex for Addition) |
| 3WiUp (100 uM) |
10uL |
| 3WiDown (100 uM) |
10uL |
| Deionized Water |
25uL |
| 10×TAE Buffer |
5uL |
