Biomod/2013/Tianjin/Experiments & Results/Cleavage

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Revision as of 20:36, 26 October 2013

Polymerizing

optimize the 1st stem-loop structure & termination

Cleavage

optimize the 2nd stem-loop structure

Delivery device

After we building the track ,we must see that if that can be cleaved by the DNAzyme, which is known as the key part of the track. In fact, this is the reason why we choose the walker with the mechanism of DNAzyme, it's easy to be characterized. We used the DNAzyme to cut the our polymer, and run a denaturation page to separate the DNA strands to see if any of them has been cut to be shorter.



At first we still set the second stem domain as 7bp. After do the polymerizing reaction, we introduce the DNAzyme into the system, also the co-factor. After performing the reaction, we run a PAGE. The double bands in both lane2 and 3 show that there’s a leakage. For the lane2 or 3, the bands means: the origin DNA, the DNA after cleavage, the DNAzyme.(In the order from the upper one) It means that the DNAzyme could cut the monomer, even it is not polymerized. This may cause the low efficiency of the walker.

Figure3.2.1 10% page. The 1st lane is a marker , just a polymer without DNAzyme, lane 2 is the monomer of 2.0nmol/L(for T1 to T4 each) with DNAzyme of 2.0nmol/L . The 3rd lane is our polymer with the origin monomer concentration as 2.0n/L, and 2.0nmol/L’s DNAzyme. The concentration of Cu2+ is 10umol/L, and Vc=100mmol/L. The band at the bottom of the lane is our DNAzyme.
Figure3.2.1 10% page. The 1st lane is a marker , just a polymer without DNAzyme, lane 2 is the monomer of 2.0nmol/L(for T1 to T4 each) with DNAzyme of 2.0nmol/L . The 3rd lane is our polymer with the origin monomer concentration as 2.0n/L, and 2.0nmol/L’s DNAzyme. The concentration of Cu2+ is 10umol/L, and Vc=100mmol/L. The band at the bottom of the lane is our DNAzyme.

So we add the length of the 2nd stem to 15bp. And the result turns out to be better. We run the gel for enough long time to separate the 2 bands, which make the band of DNAzyme can’t be seen.

Figure3.2.1 10% page. The 1st lane is a marker,a polymer without DNAzyme, lane 2 is the monomer of 2.0nmol/L(for T1 to T4 each) with DNAzyme of 2.0nmol/L . The 3rd lane is our polymer with the origin monomer concentration as 2.0n/L, and 2.0nmol/L’s DNAzyme. The concentration of Cu2+ is 10umol/L, and Vc=100mmol/L.
Figure3.2.1 10% page. The 1st lane is a marker,a polymer without DNAzyme, lane 2 is the monomer of 2.0nmol/L(for T1 to T4 each) with DNAzyme of 2.0nmol/L . The 3rd lane is our polymer with the origin monomer concentration as 2.0n/L, and 2.0nmol/L’s DNAzyme. The concentration of Cu2+ is 10umol/L, and Vc=100mmol/L.

We also detect the concentration of the cleaved and not cleaved DNA, using the florescence integration technique, the result shows that the cleaved DNA strand’s concentration is more than two folds of the DNAzymes’, which means that the multiple cleavage is happened indeed.

Figure3.2.3 The origin monomer concentration as 2.0n/L, trigger DNA=2.0nmol/L DNAzyme=2.0mol/L Cu2+ = 10umol/L, and Vc=100mmol/L. 100bp means the origin monomer, and 92bp means the DNA after cleavage.
Figure3.2.3 The origin monomer concentration as 2.0n/L, trigger DNA=2.0nmol/L DNAzyme=2.0mol/L Cu2+ = 10umol/L, and Vc=100mmol/L. 100bp means the origin monomer, and 92bp means the DNA after cleavage.




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