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| {{Komaba2014}}
| | [http://biomod2014komaba.github.io/ University of Tokyo, Komaba — UT-Komaba] |
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| #protocols{ width: 800px ;
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| <div id="protocols">
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| <h2>Protocols</h2>
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| <h3>1.Can DNA mag-beads be fixed in the wells of the microplate?</h3>
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| <h4> Determination of the ratio of b-Z-FAM and b-Z </h4>
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| <p>
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| This is done to adapt the intensity of fluorescence to the spectrophotofluorometer.<br>
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| ① The solution of b-Z and b-Z-FAM DNA is 100 μM now. So, dilute them to 1 μM. By adding 5 x SSCT buffer, we diluted them to 1 μM in 20μL. <br>
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| ② 30 μL of mag-beads were incubated and taken to a microtube. It was set on a magnetic stand for 1 minute, and supernatant fluid was dumped. We washed mag-beads three times with 60μL of 5 x SSCT buffer and added 60 μL of 5 x SSCT buffer.<br>
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| ③ The solution was divided into three equal parts. That is, mag-beads were taken to microtube for 20μL three times. Each microtube was named 1, 2 and 3. Each microtube was set on a magnetic stand for 1 minute, and supernatant fluid was dumped.<br>
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| ④ We added 0.5pmol of b-Z-FAM DNA and 4.5pmol of b-Z DNA to microtube1. So, the ratio of b-Z-FAM and b-Z in microtube1 is 1:9. In the same way, we added 1.5pmol of b-Z-FAM DNA and 3.5pmol of b-Z DNA to microtube2, and added 5pmol of b-Z-FAM DNA to microtube3. So, the ratio of b-Z-FAM and b-Z in microtube2 is 3:7, and in microtube3 is 10:0. <br>
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| ⑤ Each solution was stirred in a tube rotator for 10 minutes, and set on a magnetic stand for 1 minute, and supernatant fluid was dumped.<br>
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| ⑥ We added 20 μL of 2 x SSCT buffer to each microtube and set them on a magnetic stand for 1 minute, and supernatant fluid was dumped. <br>
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| ⑦ We added 100 μL of 5 x SSCT buffer to each microtube, and added each solution to the wells of a microplate.<br>
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| ⑧ We observed fluorescence of each well with the fluorescent microplate reader. <br>
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| </p>
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| <h3>2.Can DNA be fixed only on one hemisphere of mag-beads using photoligation? </h2>
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| <p>1. Confirmation of immobilization of DNA on the surface of mag-beads Here, we confirm that DNA can be attached to the surface of mag-beads by photoligation. We prepare mag-beads b-Z DNA fixed on. We link the end of b-Z DNA and A-cvU DNA together by using photoligation. The arrangement of Z DNA and A DNA is orthonormal, so hybridization cannot happen. We attached cA-FAM DNA by the hybridization and detect the fluorescence of the mag-beads.</p>
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| <h3>2. Immobilization of DNA on one hemisphere of mag-beads</h3>
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| <p>We immobilize A-cvU DNA on one hemisphere of beads using photoligation. We immobilize mag-beads to which b-Z DNA attached on the wells of the microplate, and link the end of b-Z and A-cvU DNA together in the same way as 2-1. Now, A DNA is immobilized only on one hemisphere of the mag-beads because photoligation occur only in the part of surface light shines on. To the wells of the plate, cA-FAM DNA is added, and the hybridization of A DNA and cA DNA happens, so fluorescent pigment is attached to the mag-beads. We detect the fluorescence of the mag-beads to know if A DNA attaches only on one hemisphere.</p>
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| </body>
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