Design

The following figures show the sequence motif of the DNA and their nucleotide sequences. The small letters of a, b, c, … mean each motif, and letters with prime mean complementary sequences.

Pre-simulation

Using the NUPACK(http://www.nupack.org/), the software for the analysis and the design of sequences, the states of the combinations of the above sequences was calculated.

Figure 1. Similation results of the combination of H1, H2 and I_H.
link to nupack

Figure 2. Similation results of the combination of A1 and A2.
link to nupack

Figure 3. Similation results of the combination of A1, A2 and C.
link to nupack

Figure 4. Similation results of the combination of A1, A2, C and C'.
link to nupack

Figure 5. Similation results of the combination of A3 and A4.
link to nupack

Protocol

1. Electrophoresis of st1 and I_H

The states of the combinations were estimated based on the result of agarose gel electrophoresis. The run time of electrophoresis was 2 hours.

Table 1. Summary of the experiment 1 condition. The final concentration of the each solution is shown in the table.
Sample index 1 2 3
TE(pH=8.0) 7 8 8
NaCl(1500mM) 1 1 1
I_H(10µM) 1 0 1
st1(10µM) 1 1 0
Loading Buffer 2 2 2
Total(µL) 12 12 12

2. Electrophoresis of combination H1, H2 and I_H

The states of the combinations were estimated based on the result of electrophoresis.

Table 2. Summary of the experiment 2 condition. The final concentration of the each solution is shown in the table.
Sample index 1 2 3 4 5 6 7
TE(pH=8.0) 7.9 7.9 7.9 7.9 7.9 7.9 7.9
NaCl(5M) 0.3 0.3 0.3 0.3 0.3 0.3 0.3
H1(10µM) 0.6 0.6 0 0.6 0 0.6 0
H2(10µM) 0.6 0.6 0.6 0 0.6 0 0
I_H(10µM) 0.6 0 0.6 0.6 0 0 0.6
Loading Buffer 2 2 2 2 2 2 2
Water 0 0.6 0.6 0.6 1.2 1.2 1.2
Total(µL) 12 12 12 12 12 12 12

3. Melting curve with change of [I_H]

Based on the melting curve, the change of the ratio of double-strand was measured along the temperature fall, and the effect of concentration of I_H on the formation of the gels was estimated. The DNA strands were first kept at 90℃ for 3 minutes and then the temperature was lowered to 20℃ by 1℃ per minute.

Table 3. Summary of the experiment 3 condition. The final concentration of the each solution is shown in the table.
Sample index 1 2 3 4 5
TE(pH=8.0) 3.6 3.6 3.6 3.6 3.6
H1(1mM) 0.8 0.8 0.8 0.8 0.8
H2(1mM) 0.8 0.8 0.8 0.8 0.8
NaCl(500mM) 1.6 1.6 1.6 1.6 1.6
EvaGreen 0.4 0.4 0.4 0.4 0.4
Beacon 0.8 0.8 0.8 0.8 0.8
I_H 0 0.5 (100µM) 1 (100µM) 2 (100µM) 0.5 (1mM)
Water 2 1.5 1 0 1.5
Total(µL) 10 10 10 10 10

4. Melting curve with change of [Na+]

Based on the melting curve, the change of the ratio of double-strand was measured along the temperature fall, and the effect of NaCl on the formation of the gels was estimated. This time, the DNA strands were kept at 95℃ for 10 minutes and then the temperature was lowered to 25℃ by 1℃ per 50 seconds.

Table 4. Summary of the experiment 4 condition. The final concentration of the each solution is shown in the table.
Sample index 1 2 3 4
TE(pH=8.0) 4.5 4.5 4.5 4.5
H1(1mM) 1 1 1 1
H2(1mM) 1 1 1 1
I_H(1mM) 1 1 1 1
EvaGreen 0.5 0.5 0.5 0.5
NaCl 2 (125mM) 2 (250mM) 2 (500mM) 2 (1000mM)
Total(µL) 10 10 10 10

5. Melting curve with change of [Mg2+]

Based on the melting curve, the effect of the concentration of Mg2+ on the formation of the gels was estimated. The annealing procedure was as same as experiment 3.

Table 5. Summary of the experiment 5 condition. The final concentration of the each solution is shown in the table.
Sample index 1 2 3 4
TE(pH=8.0) 4.5 4.5 4.5 4.5
EvaGreen 0.5 0.5 0.5 0.5
H1(1mM) 1 1 1 1
H2(1mM) 1 1 1 1
I_H(100µM) 1 1 1 1
MgSO4(100mM) 0 0.5 1 2
Loading Buffer 2 2 2 2
Water 2 1.5 1 0
Total(µL) 10 10 10 10

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