Biomod/2014/Komaba/Protocols
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Protocols
1.Can DNA mag-beads be fixed in the wells of the microplate?
Determination of the ratio of b-Z-FAM and b-Z
This is done to adapt the intensity of fluorescence to the spectrophotofluorometer.
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.
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.
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.
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.
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.
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.
⑦ We added 100 μL of 5 x SSCT buffer to each microtube, and added each solution to the wells of a microplate.
⑧ We observed fluorescence of each well with the fluorescent microplate reader.
2.Can DNA be fixed only on one hemisphere of mag-beads using photoligation?
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.
2. Immobilization of DNA on one hemisphere of mag-beads
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 hybridization3. Can mag-beads be fixed in the wells of the microplate turning upside down?
3. Can mag-beads be fixed in the wells of the microplate turning upside down?
In step 1 and 2, DNA immobilized in the wells of plate was mainly b-cZ DNA. However, from here, we immobilize b-cA DNA. Now, A-DNA is attached only on one hemisphere of the mag-beads, and on the other hemisphere, Z-DNA is attached. So, when we add the mag-beads to the cA-immobilized well, the beads will be immobilized in the well turning upside down. 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.
4. Can DNA be fixed on the other hemisphere of mag-beads using photoligation?
We fix another DNA (B-cvU) on the other hemisphere of mag-beads in the same way as step2. Now, A DNA is fixed on the north hemisphere of the beads, and B DNA is on the south hemisphere. We add cB-Texasred DNA to the wells of the microplate, and the hybridization between B and cB DNA will occur. We observe the intensity of the fluorescence if Texasred is on the surface of the mag-beads.
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