Biomod/2014/Komaba/Protocols
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<a class="navbar-brand" href="http://openwetware.org/wiki/Biomod/2014/Komaba">Komaba</a>
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<li><a href="http://openwetware.org/wiki/Biomod/2014/Komaba/Protocols">Protocols</a></li>
<li><a href="http://openwetware.org/wiki/Biomod/2014/Komaba/Equipment">Equipment</a></li>
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</li> <li><a href="http://openwetware.org/wiki/Biomod/2014/Komaba/Results">Results</a></li>
<li><a href="http://openwetware.org/wiki/Biomod/2014/Komaba/Team">Team</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2014/Komaba/Acknowledgement">Acknowledgement</a></li>
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Protocols
1.Can DNA mag-beads be fixed in the wells of the microplate?
1. 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.
<a href="http://openwetware.org/wiki/Biomod/2014/Komaba/Results#result11">result</a>
2. Confirmation of the specific bonding between wells of a microplate and mag-beads
Here, we prepared three kinds of wells, b-cZ DNA was fixed on, b-cA was fixed on, and nothing was fixed on. We added beads that b-Z-FAM DNA was fixed on to each kind of well. Each kind of well has the well to which added one layer of beads and five layers of beads. We expected that beads would bond only with the wells b-cZ DNA fixed on because the beads could fix on the wells only by base complementarity.
We detected the intensity of fluorescence of each well. If fluorescence can be detected in the well with b-cZ and cannot be detected in the well with b-cA and with nothing, the specific bonding between the well with b-Z and beads with Z DNA is confirmed.
① By adding 5 x SSCT buffer, we diluted b-cZ and b-cA DNA to 1μ
M in 500μL, b-Z-FAM DNA to 1μM in 300μL.
② We pre-washed wells of plate three times with 300μL/well 5 x SSCT buffer without any incubation step.
③
Picture 2
<img src="http://openwetware.org/images/3/34/Result121.PNG">
④ Each well of a microplate was used like this figure. Into the cZ and cA well, we added 50μL of 1μM of the solution of b-cZ and b-cA DNA, and added 50μL of 5 x SSCT buffer. Into the Nothing and Blank well, we added 100μL of 5 x SSCT buffer. Now, in the cZ and cA well, there is 50 pmol of DNA.
⑤ The plate was incubated with gentle agitation for 1 hour at room temperature.
⑥ We began to prepare beads while incubating the plate. 180μL of mag-beads were taken to a microtube. It was set on a magnet stand for 1 minute and supernatant fluid was dumped.
⑦ Beads were washed three times with 360 μL of 5 x SSCT buffer.
⑧ To the microtube, 90 μL of b-Z-FAM DNA and 90 μL of 5 x
SSCT buffer were added. The solution was stirred for 10 minutes. The microtube was set on a magnet stand for 1 minute and
supernatant fluid was dumped. Now, there was 90 pmol of b-Z-FAM DNA in the microtube. DNA was fixed on the beads.
⑨ 180 μL of 5 x SSCT buffer was added to the microtube. We separated the solution into two parts. One is 150 μL, that is, 3 x 5 layers of a well of the plate of mag-beads (3 x 25 pmol), and the other is 30 μL, that is, 3 x 1 layer (3 x 5 pmol). We named the former 1, and the latter 2. Each tube was set on a magnet for 1 minute, and supernatant fluid was dumped.
⑩ We added 300μL of 2 x SSCT buffer to the microtube 1, set on a magnet stand for 1 minute, and dumped the supernatant fluid. In similar way, we added 60 μL of 2 x SSCT buffer to the microtube 2, and set on a magnet stand for 1 minute, and dumped the supernatant fluid. This step was repeated three times.
⑪ 300μL of 5 x SSCT buffer was added to each microtube.
⑫ We stopped agitation of the microplate and washed the wells of the plate three times with 300μL/well of 2 x SSCT buffer.
⑬ Mag-beads were added to the wells of the plate. We added to the cZ, cA, and nothing well in picture 2. To the upper cZ,cA, and nothing well, 100μL/well of the solution in the microtube1 was added. To the bottom cZ,cA, and nothing well, 100μL/well of the solution in the microtube2 was added. To the blank well, 100μ L/well of 5 x SSCT buffer was added.
⑭ The plate was incubated for 10 minutes at room temperature. While this time, the hybridization between Z DNA and cZ DNA proceeds. The supernatant fluid was dumped.
⑮ We washed each well of the plate with 300 μL/well of 2 x SSCT buffer three times.
⑯ 100μL/well of 5 x SSCT buffer was added to each well.
⑰ We observed fluorescence of each well with the fluorescent microplate reader.
<a href="http://openwetware.org/wiki/Biomod/2014/Komaba/Results#result12">result</a>
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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