Team:DUT China A/Lab Book
September 14th
1. Hybridization the circular template the primer:
Add 160ul of Aptamer-Primer 1 (200nm) annealing 5 min at 95℃ and cool to 20℃. Then add it to 7 wells, each well for 20ul, in turn marked A, B1, B2, B3, C1, C2, C3, D. Add 20ul circular template, 80ul TE buffer per well, mix and incubate them at room temperature for 2 hours.2. The R-Process
Add 100ul of reaction buffer to A, B1, B2, B3, D, 50ul reaction buffer in C1, C2, C3, and 50ul of reaction buffer in C1, C2, C3; then add 50ul of dNTP(25mM) per well except A, add 0.5 ul of φ29 per well except D, then incubate at room temperature.3. The M-Process
After incubating 40min, add 1ul of primer 2 and 3 to the well A, B1, C1, D , 1ul, and put in the thermostat to continue incubate. After 80min, add 1 ul of primer 2 and 3 to the well B2, C2 ,then put in the thermostat to continue incubate. After 120min, add 1 ul of primer 2 and 3 to the well B3, C3, then put in the thermostat to continue incubate.Result:
We use the enzyme-labeled instrument to test the absorbance of the sample to observe the formation of hydrogels. However, it was found that there was no excessive change in the absorbent value, so it is not feasible to observe hydrogel formation in real time using the enzyme-labeled instrument.September 23th
1. Hybridization the circular template the primer:
Add 50 ul of Aptamer-Primer 1 (200nM) to a centrifuge tube, annealing 5min at 95℃, and cool to 20℃. Take tube A, B, C, then add 40ul 1*TE (10mM Tris-HCl, 1mM EDTA PH=8.0) solution in tube B、C , add 40ul ddH2O in tube A, and then add 10ul of circular template and 10ul of Aptamer-Primer 1, mix and incubate for 2 hours in a 30-degree incubator.2. The R-Process
Add 50ul of 10*phi 29 buffer (330mM Tris-acetate, 660mM K- acetate, 100 mM Mg- acetate, 1% Tween 20, 10mM DTT, PH=7.9）to tube A、B, add 50ul of 10*phi29 buffer (which was purchased from New England Biolabs) to the tube C , and add 10ul of dNTP mixer (25mM) and 1ul of phi29 DNA polymerase (10U/ul) per tube. After that, incubate for 45 min at 30 ℃.3. The M-Process
Add 1 ul of primer 2 and 3(100uM) to each tube and continue to incubate at 30 ° C for 16 hours.September 24th
Hydrogels observation 1:
There is no obvious phenomenon in tube A; tube B is more turbid than A, gels were too sticky so they were pulled out as silk when we are pipetting them; tube C is more turbid than A, which is not as good as B and the gel were also sticky.Analysis:
(1) The addition of TE solution is a necessary condition for hydrogel formation.(2) On the basis of the same sample volume, the homemade phi29 buffer is better for this capacity system.
Hydrogels observation 2:
Formulated 3% agaric gel for electrophoresis, added the circular template, products of tube A, tube B and tube C to the sample port respectively.Result:
There was no phenomenon in the hole which added circular template, and gels were difficult to migrate through the agarose gel and remained the retention in home position for the remaining three holes.September 26th
Hydrogels observation 3:
Test B-tube samples with a scanning electron microscope.Result:
The glues were found with a diameter of about 1-2 nm.Analysis:
Hydrogel preparation in tube B was preliminary successful.September 27th
1. Hybridization the circular template the primer:
Add 50 ul of Aptamer-Primer 1 (200nM) to a centrifuge tube, annealing 5min at 95℃, and cool to 20℃.Take two centrifuge tubes and marked with X、Y. Add 10ul circular template, 10 ul of Aptamer-Primer 1 (200nM) and 40ul 1*TE buffer per tube, mix and incubate them at room temperature for 2 hours.2. The R-Process
Add 50ul of 10*phi 29 buffer (330mM Tris-acetate, 660mM K- acetate, 100 mM Mg- acetate, 1% Tween 20, 10mM DTT, PH=7.9）and 10ul of dNTP mixer (25mM) to tube X、Y, and add 1ul of phi29 DNA polymerase (10U/ul) to tube Y and 1 ul of ddH2O to tube X. After that, incubate for 45 min at 30 ℃.3. The M-Process
Add 1 ul of primer 2 and 3(100uM) to each tube and continue to incubate at 30 ° C for 16 hours.September 29th
1. Add 100ul of Aptamer-Primer 1 (200nM) in a centrifuge tube, annealing 5 min at 95℃, and cool to 20℃.
2. Add 10ul of Aptamer-Primer 1, 10ul of circular template, 30ul of TE buffer (pH = 8.0) to three centrifuge tubes named 1,2,3, then incubate at 30 ℃ for 2h.
3. Add 50ul of 10*phi 29 reaction buffer (pH=7.9), 1.5ul of phi 29 DNA polymerase(10U/ul) and 20ul dNTP mixer (25mM) to tube 1.
Add 50ul of 1*phi 29 reaction buffer (pH=7.9), 1.5ul of phi 29 DNA polymerase(10U/ul) and 20ul dNTP mixer (25mM) to tube 2.
Add 50ul of 1*phi 29 reaction buffer (pH=7.9) and 20ul dNTP mixer (25mM) to tube 3.
Incubate them at 30 ℃ for 50min.
4. Add 1.5ul of Primer 2 (100uM), 1.5uL Primer 3 (100uM) to tube 1,2,3, incubate at 30 ℃ for 16h and observe every 2h.
5. Take 100ul from tube 1,2,3, and dilute to 2 ml. Measure the size of the hydrogel with Laser Particle Size Analyzer.
Result
Analyze
The preparation of hydrogel is initially successful, and further optimization of experimental conditions is needed.October 2nd
1. Before the experiment, we first mixed several tubes of circular template into a tube, and compound 10*phi29 reaction buffer (330mM Tris-acetate, 660mM K- acetate, 100 mM Mg- acetate, 1% Tween 20, 10mM DTT), then dilute to 3* phi29 reaction buffer.
2. Add 100ul of Aptamer-Primer 1 (200nM) in a centrifuge tube, annealing 5 min at 95℃, and cool to 20℃.
3. Add 10ul of Aptamer-Primer 1, 10ul of circular template, and 30ul of TE buffer (pH = 8.0) to three centrifuge tubes named 1,2,3, then incubate at 30 ℃ for 2h.
4. Add 50ul of 10*phi 29 reaction buffer (pH=7.9), 1.5ul of phi 29 DNA polymerase(10U/ul) and 20ul dNTP mixer (25mM) to tube 1.
Add 50ul of 3*phi 29 reaction buffer (pH=7.9), 1.5ul of phi 29 DNA polymerase(10U/ul) and 20ul dNTP mixer (25mM) to tube 2.
Add 50ul of 3*phi 29 reaction buffer (pH=7.9), 1.5ul of phi 29 DNA polymerase(10U/ul) and 20ul dNTP mixer (25mM) to tube 3.
Incubate them at 30 ℃ for 50min.
5. Add 1.5ul of Primer 2 (100uM), 1.5uL Primer 3 (100uM) to tube 1,2,3, incubate at 30 ℃ for 16h and observe every 2h.
Results
Gels from tube 1 and tube 2 were too sticky so they were pulled out as silk when we are pipetting them. After centrifugation for 10 minutes at 10000 revolutions, transparent colloid is obtained in tube 2.
Analyze
The experimental conditions of the tube 2 is suitable.October 3rd
1. Add 20ul of Aptamer-Primer 1 (200nM) in a centrifuge tube, annealing 5 min at 95℃, and cool to 20℃.
2. Add 10ul of Aptamer-Primer 1 named 1,2, 10ul of circular template and 30ul of TE buffer (pH = 8.0) to two centrifuge tubes and incubate at 30 ℃ for 2h.
3. Add 50ul of 2.4*phi 29 reaction buffer (pH=7.9), 1.5ul of phi 29 DNA polymerase(10U/ul) and 20ul dNTP mixer (25mM) to tube 1.
Add 50ul of 3*phi 29 reaction buffer (pH=7.9), 1.5ul of phi 29 DNA polymerase(10U/ul) and 20ul dNTP mixer (25mM) to tube 2.
Incubate them at 30 ℃ for 40min.
4. Add 1.5ul of Primer 2 (100uM), 1.5uL Primer 3 (100uM) to tube 1,2, incubate at 30 ℃ for 16h and observe every 2h.
Results
Gels from tube 2 were too sticky so they were pulled out as silk when we are pipetting them, and no obvious phenomenon is found in tube 1.
After 1% agarose gel electrophoresis, result is listed below:
Figure
(1) DL500 DNA marker (2) DL2000 DNA marker (3) sample from tube 1 (4) sample from tube 2Analyze
Lane 3 is tube 1, apparently there is no gelation; although lane 4 has gelatinized, there is still brighter bands below, and we considered that lots of the long chain of Ap3 which have already after the end of R process still has not entered the next stage, which may be because of the sort of the primer2 and 3, the next experiment will increase the concentration of both.October 4th
1. Add 20ul of Aptamer-Primer 1 (200nM) in a centrifuge tube, annealing 5 min at 95℃, and cool to 20℃.
2. Add 10ul of Aptamer-Primer 1, 10ul of circular template and 30ul of TE buffer (pH = 8.0) to two centrifuge tubes named Y, N.
Add 10ul of Aptamer-Primer 1, 10ul of circular template and 30ul of TE buffer (pH = 8.0) into two wells named 1,2.
Incubate them at 30 ℃ for 2h.
3. Add 50ul of 3*phi 29 reaction buffer (pH=7.9), 1.5ul of phi 29 DNA polymerase(10U/ul) and 20ul dNTP mixer (25mM) to tube Y.
Add 50ul of 3*phi 29 reaction buffer (pH=7.9) and 20ul dNTP mixer (25mM) to tube N.
Add 50ul of 3* phi 29 reaction buffer (pH=7.4) and 20ul of dNTP mixer (25mM) to the two wells of 96-well plate, then add 1.5ul of phi 29 DNA polymerase(10U/ul) to well 1.
4. Add 1.5ul of Primer 2 (100uM), 1.5uL Primer 3 (100uM) to tube 1,2 and the well 1,2, incubate at 30 ℃ for 16h and observe every 2h.
Results
After 16h, no obvious phenomenon is found in the group with the buffer (pH=7.4) After 72h, Gels in the group were too sticky so they were pulled out as silk when we are pipetting them.
October 7th
1. Add 20ul of Aptamer-Primer 1 (200nM) in a centrifuge tube, annealing 5 min at 95℃, and cool to 20℃.
2. Add 10ul of Aptamer-Primer 1, 10ul of circular template and 30ul of TE buffer (pH = 7.5) to two centrifuge tubes named Y, N, incubate at 30 ℃ for 2h.
3. Add 50ul of 3*phi 29 reaction buffer (pH=7.9), 1.5ul of phi 29 DNA polymerase(10U/ul) and 20ul dNTP mixer (25mM) to tube Y.
Add 50ul of 3*phi 29 reaction buffer (pH=7.9) and 20ul dNTP mixer (25mM) to tube N.
Incubate them at 30 ℃ for 40min.
4. Add 5ul of Primer 2 (100uM), 5uL Primer 3 (100uM) to tube Y, N, then incubate at 30 ℃ for 16h and observe every 2h.
Result
Gels from tube Y were too sticky so they were pulled out as silk when we are pipetting them, and its formation was faster.Analyze
More primer 2 and 3 can be added to increase the amount of glue and shorten the formation time.October 13th
1. Add 20 ul of Aptamer-Primer 1 (200nM) to a centrifuge tube, annealing 5min at 95℃, and cool to 20℃.
2. Take two centrifuge tubes named Y, N. Add 10ul of circular template, 10 ul of Aptamer-Primer 1 (200nM) and 30ul TE buffer(pH=7.5) per tube, mix and incubate at 30 ℃ for 2h.
3. Add 8ul of 10*phi 29 buffer (330mM Tris-acetate, 660mM K- acetate, 100 mM Mg- acetate, 1% Tween 20, 10mM DTT, PH=7.4) , 20ul of dNTP mixer (25mM) and 0.4ul of BSA to tube 1、2, and add 1ul of phi29 DNA polymerase (10U/ul) to tube 1 and 1 ul of ddH2O to tube 2, then incubate for 45 min at 30 ℃.
4. Add 5ul of Primer 2 (100uM), 5uL Primer 3 (100uM) to tube Y, N, then incubate at 30 ℃ for 16h and observe every 2h.
October 15th
1. Add 20 ul of Aptamer-Primer 1 (200nM) to a centrifuge tube, annealing 5min at 95℃, and cool to 20℃.
2. Take two centrifuge tubes named Y, N. Add 10ul of circular template, 10 ul of Aptamer-Primer 1 (200nM) and 30ul TE buffer(pH=7.5) per tube, mix and incubate at 30 ℃ for 2h.
3. Add 8ul of 10*phi 29 buffer (330mM Tris-acetate, 660mM K- acetate, 100 mM Mg- acetate, 1% Tween 20, 10mM DTT, PH=7.4) , 20ul of dNTP mixer (25mM) and 0.4ul of BSA to tube 1、2, and add 1.5ul of phi29 DNA polymerase (10U/ul) to tube 1 and 1 ul of ddH2O to tube 2, then incubate for 45 min at 30 ℃.
4. Add 5ul of Primer 2 (100uM), 5uL Primer 3 (100uM) to tube Y, N, then incubate at 30 ℃ for 16h and observe every 2h.
October 15th
1. Add 20 ul of Aptamer-Primer 1 (200nM) to a centrifuge tube, annealing 5min at 95℃, and cool to 20℃.
2. Take two centrifuge tubes named Y, N. Add 10ul of circular template, 10 ul of Aptamer-Primer 1 (200nM) and 30ul TE buffer(pH=7.5) per tube, mix and incubate at 30 ℃ for 2h.
3. Add 8ul of 10*phi 29 buffer (330mM Tris-acetate, 660mM K- acetate, 100 mM Mg- acetate, 1% Tween 20, 10mM DTT, PH=7.4) , 20ul of dNTP mixer (25mM) and 0.4ul of BSA to tube 1、2, and add 1.5ul of phi29 DNA polymerase (10U/ul) to tube 1 and 1 ul of ddH2O to tube 2, then incubate for 45 min at 30 ℃.
4. Add 5ul of Primer 2 (100uM), 5uL Primer 3 (100uM) to tube Y, N, then incubate at 30 ℃ for 16h and observe every 2h.