基礎ゼミチーム/basic seminar team/experiment result

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BIOMOD2012 Tohoku Team B




Experiment


2012/09/04
<img src="http://openwetware.org/images/thumb/2/27/20120904%2C100v%2C2h%2Cfrige.jpg/708px-20120904%2C100v%2C2h%2Cfrige.jpg" style="width:500px;float:left;margin:5px;">
condition
constant voltage 50V
temparature:4℃(in refrigerate)
the electrophoresis for 2 hours in a refrigerator
SYBR Gold as a stain for 10 minutes
1X TAE added to Mg2+ as a buffer
result
The result was what we wanted completely.
Target DNA hibridized to Selector 1
Target DNA moved from Selector 1 to Selector2, and Selector 2 to Selector 3


2012/08/30
<img src="http://openwetware.org/images/thumb/c/c7/20120830_%E2%91%A1.jpg/800px-20120830_%E2%91%A1.jpg" style="width:500px;float:left;margin:5px;">
condition
constant voltage 50V
1X TAE added to Mg2+ as a buffer
The room temperature was 33℃(4℃ in the refrigerator)
the electrophoresis for 3.5 hours in a refrigerator
SYBR Gold as a stain for 10 minutes
result
The result was what we had expected.
Finally, Selector 1 hybridized with the target.


2012/08/29
<img src="http://openwetware.org/images/thumb/4/4f/20120829_%E2%91%A1.jpg/800px-20120829_%E2%91%A1.jpg" style="width:500px;float:left;margin:5px;">
condition
1X TAE added to Mg2+ as a buffer
The room temperature was 33℃ (4℃ in the refrigerator)
the electrophoresis for 3.5 hours
SYBR Gold as a stain for 10 minutes
result
Selector 1 didn't hybridize with target. But the Other samples worked well.
We will try again with cool buffer tomorrow.


2012/08/28
<img src="http://openwetware.org/images/thumb/5/5a/20120828_%E2%91%A1.jpg/800px-20120828_%E2%91%A1.jpg" style="width:500px;float:left;margin:5px;">
condition
constant voltage 50V
1X TAE was added to Mg2+ as a buffer
The room temperature was 33℃
the electrophoresis for 3 hours
SYBR Gold as a stain for 10 minutes
result
We used the improved design DNA. Selector 1 didn't hybridize with the target, but the other results were what we had expected.
The melting temperature of Selector 1 was lower than today's room temperature. And as for Selector 1, no hybridization occurred.
Tomorrow we will do the experiment in a refrigerator.


2012/08/24
<img src="http://openwetware.org/images/thumb/a/af/20120824_%E2%91%A1.jpg/791px-20120824_%E2%91%A1.jpg" style="width:500px;float:left;margin:5px;">
condition
constant voltage 50V
1X TAE was added to Mg2+ as a buffer
The room temperature was 28℃
the electrophoresis for 3 hours
SYBR Gold as a stain for 10 minutes
result
Selector 1 seems to hybridize with itself.


2012/08/22
<img src="http://openwetware.org/images/thumb/e/e3/20120822_%E2%91%A1.jpg/800px-20120822_%E2%91%A1.jpg" style="width:500px;float:left;margin:5px;">
condition
constant voltage 50V
1X TAE was added to Mg2+ as a buffer
The room temperature was 28℃
the electrophoresis for 3 hours
SYBR Gold as a stain for 10 minutes
result
The target and Selector 1, and the target and Selector 2, seemed to hybridize.
In the lane of Selector 1 and Selector 3, single-stranded Selector 1 and Selector 3 were seen. So Selector 1 and Selector 3 don't interact with each other.
In the lane of Selector 2 and Selector 3, We could see a black band at one place.
Maybe Selector 2's and Selector3's hairpin structure interacted with each other.



2012/08/21
<img src="http://openwetware.org/images/thumb/b/be/20120821_%E2%91%A1.jpg/800px-20120821_%E2%91%A1.jpg" style="width:500px;float:left;margin:5px;">
condition
constant voltage 50V
1X TAE was added to Mg2+ as a buffer
The room temperature was 28℃
the electrophoresis for 3 hours
SYBR Gold as a stain for 10 minutes
We did electrophoresis at 50V in a fridge (at 4 ℃)
To get the target to move between Selectors, we waited one hour before the next step
result
As a result, in the lane of the target, Selector 1, and 2, the band of Selector 1 couldn't be seen. And the band of Selector 2 was seen at the same place as when it was single-stranded.
The target didn't seem to attach either to Selector 1 or 2.
So Selector 1 and 2 aggregated with themselves or each other.
As the 20% gel is difficult to see the result and it takes a long time, We'll use only the 10% gel from now on.


2012/08/20
<img src="http://openwetware.org/images/thumb/5/5d/20120820_10-_%E2%91%A1.jpg/800px-20120820_10-_%E2%91%A1.jpg" style="width:500px;float:left;margin:5px;">
<img src="http://openwetware.org/images/thumb/a/af/201120820_20-_%E2%91%A1.jpg/767px-201120820_20-_%E2%91%A1.jpg" style="width:500px;float:left;margin:5px;">
condition
constant voltage 50V
1X TAE was added to Mg2+ as a buffer
The room temperature was 28℃
the electrophoresis for 3 hours
SYBR Gold as a stain for 10 minutes
We did electrophoresis at 50V in a fridge (at 4 ℃)
To get the target to move between Selectors, we waited one hour before the next step
result
The target hybridized with Selector 2 in the lane of the target, Selector 1, and 2. The target also hybridized with Selector 3 in the lane of the target, Selector 1, 2, and 3.
From this result, it's possible that Target DNA moved from Selector 1 to Selecor 2, and from Selector 2 to Selector 3.
However, in the lane of the target, Selector 1 and 2, the band of the target and Selector 1 was also clear. So we cannot decide the target actually moved from Selector 1 to Selector 2.
In the lane of the target, Selector 1, 2, and 3, no band was around the place of single-stranded Selector 1. Probably we forgot to put Selector 1.
Today we don't make sure the target moved from Selector 1 to Selector 2. But it's likely the target moved from Selector 2 to Selector 3.


2012/08/17
<img src="http://openwetware.org/images/thumb/e/e3/20120817_%E2%91%A1.jpg/800px-20120817_%E2%91%A1.jpg" style="width:500px;float:left;margin:5px;">
condition
constant voltage 100V
1X TAE as a buffer
The room temperature was 28℃
SYBR Gold as a stain for 10 minutes
the electrophoresisin a fridge (at 4 ℃)
To get the target to move between Selectors, we waited 50 minutes before the next step
result
The materials diffused and the bands weren't clear, because we forgot to put the 15μl buffer of 1.25 % concentration of Mg2+ to samples.


2012/08/09
<img src="http://openwetware.org/images/thumb/e/e3/20120809_%E2%91%A1.jpg/800px-20120809_%E2%91%A1.jpg" style="width:500px;float:left;margin:5px;">
condition
1X TAE as a buffer
The room temperature was 28℃
SYBR Gold as a stain for 10 minutes
We did electrophoresis at constant voltage 100V in a fridge (at 4 ℃)
To get the target to move between Selectors, we waited 30 minutes before the next step
result
We used 10% and 20 % acrylamide gel for the electrophoresis.
We stained the 10% gel with midori green, but the gel wasn't stained clearly(the picture is not here).
The above picture is the 20% gel stained with SYBR gold.






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