Method for cutting DNA based on flax cyclopeptide A
A cutting method and cyclic peptide technology, applied in the direction of peptides, hydrolases, etc., can solve problems such as unseen interactions
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Embodiment 1
[0045] Example 1: Optimal reaction time for cleaving DNA with cyclic peptide A
[0046] The optimal reaction time for cleaving DNA by cyclic peptide A was detected by gel electrophoresis.
[0047] Choose Mg 2+ As the auxiliary ion, the BR buffer with pH 7.0 was used as the reaction system, and the cyclic peptide A concentration of 25 mg / L was used as the reaction concentration. The optimal reaction time for cyclic peptide A to cut DNA was screened out by reacting for different times.
[0048] (1) Preparation of sample: add to 1.5mL EP tube, add 15.6μL of BR buffer solution, 2μL of target compound solution, 2μL of metal ion solution in sequence, mix thoroughly and evenly by vortex mixer, after centrifugation Add 0.4 μL of 0.5 mg / mL pUC19 DNA plasmid, mark the numbers, mix well and then centrifuge with a centrifuge, seal and place in a 37°C incubator to react for different times.
[0049](2) Preparation of 1% agarose gel: Weigh 1 g of agarose into a 200 mL beaker filled with 1...
Embodiment 2
[0055] Example 2: The optimal auxiliary metal ion for cleaving DNA by cyclic peptide A
[0056] The effect of ion species on DNA cleavage by cyclic peptide A was examined using gel electrophoresis.
[0057] Four divalent metal ions were selected as auxiliary ions, namely Mg 2+ , Cu 2+ , Zn 2+ and Ca 2+ .
[0058] (1) Preparation of sample: add to 1.5mL EP tube, add 15.6μL of BR buffer solution with pH value of 7.0, 2μL of target compound solution, 2μL of metal ion solution in sequence, and mix thoroughly by vortex mixer Uniformly, after centrifugation, add 0.4 μL of 0.5 mg / mL pUC19 DNA plasmid, make a number mark, mix well and then centrifuge with a centrifuge, seal and place in a 37°C incubator for 24 hours.
[0059] (2) Preparation of 1% agarose gel: Weigh 1 g of agarose into a 200 mL beaker filled with 100 mL of 1×TAE solution, heat and shake it repeatedly in a microwave oven to completely dissolve until the solution is transparent. As for cooling below 60°C at room t...
Embodiment 3
[0065] Example 3: The optimal concentration of auxiliary metal ions for cleaving DNA by cyclic peptide A
[0066] As can be seen from Example 2, Mg 2+ It is the best auxiliary metal ion for cyclic peptide A to cleave DNA, so as to explore the optimal ion concentration.
[0067] Choose Mg 2+ The concentrations are 100mM, 50mM, 10mM, 5mM and 1mM, respectively.
[0068] (1) Preparation of sample: add to 1.5mL EP tube, add 15.6μL of BR buffer solution with a pH value of 7.0, 2μL of target compound solution, and 2μL of metal ion solution in sequence, and make it fully through the vortex mixer Mix evenly, add 0.4 μL of 0.5 mg / mL pUC19 DNA plasmid after centrifugation, mark the numbers, mix evenly and then centrifuge with a centrifuge, seal and place in a 37°C incubator for 24 hours.
[0069] (2) Preparation of 1% agarose gel: Weigh 1 g of agarose into a 200 mL beaker filled with 100 mL of 1×TAE solution, heat and shake it repeatedly in a microwave oven to completely dissolve unti...
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