Team:KIT-Kyoto/Notebook-week3

From 2012.igem.org

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<h2>August 20th</h2>
<h2>August 20th</h2>
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<strong>TNFA and API2</strong>
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<br><br>
1. Reproduction of DNA from gel<br>
1. Reproduction of DNA from gel<br>
   We did agarose gel electrophoresis (0.7% gel).<br><br>
   We did agarose gel electrophoresis (0.7% gel).<br><br>
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<h2>August 21st</h2>
<h2>August 21st</h2>
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<strong>TNFA and API2</strong>
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<br><br>
1. Density measurement of attB TNFAIP3<br>
1. Density measurement of attB TNFAIP3<br>
  We did agarose gel electrophoresis (0.7% gel). (1kb marker-3uL, 5times dilution attB TNFAIP3-1uL (make August 20 ), 10times dilution-1uL, 1kb marker-2uL, 15times dilution-1uL, 20times dilution-1uL, 1kb marker-1uL )<br><br>
  We did agarose gel electrophoresis (0.7% gel). (1kb marker-3uL, 5times dilution attB TNFAIP3-1uL (make August 20 ), 10times dilution-1uL, 1kb marker-2uL, 15times dilution-1uL, 20times dilution-1uL, 1kb marker-1uL )<br><br>
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<h2>August 22nd</h2>
<h2>August 22nd</h2>
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<strong>TNFA and API2</strong>
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<br><br>
We separated six colonies from 40uL spread plate of BP TNFAIP3 (make August 21 ) .<br>
We separated six colonies from 40uL spread plate of BP TNFAIP3 (make August 21 ) .<br>
We cultivated these colonies on 2.5mL Kanamycin(+) LB culture medium and we kept shaking this medium overnight.<br>
We cultivated these colonies on 2.5mL Kanamycin(+) LB culture medium and we kept shaking this medium overnight.<br>
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<h2>August 23rd</h2>
<h2>August 23rd</h2>
<br>
<br>
 +
<strong>TNFA and API2</strong>
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<br><br>
<img src="http://2012.igem.org/wiki/images/8/81/0823.png" width="500" height="300">
<img src="http://2012.igem.org/wiki/images/8/81/0823.png" width="500" height="300">
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Revision as of 10:42, 23 September 2012






August 20th


TNFA and API2

1. Reproduction of DNA from gel
We did agarose gel electrophoresis (0.7% gel).

Composition
a product of PCR attB TNFAIP3(8/1)
DNA sample90μL
6×Dye18μL
Total108μL

We separated 54uL×2 from 108uL (sample) and we did electrophoresis (54uL sample and 1kb marker 5uL).We separated gel containing DNA.

Results



We isolated DNA from these gels by QIA quick Gel Extraction Kit.
Finally we melt DNA to TE Buffer(40uL)


August 21st


TNFA and API2

1. Density measurement of attB TNFAIP3
We did agarose gel electrophoresis (0.7% gel). (1kb marker-3uL, 5times dilution attB TNFAIP3-1uL (make August 20 ), 10times dilution-1uL, 1kb marker-2uL, 15times dilution-1uL, 20times dilution-1uL, 1kb marker-1uL )

Results



We estimated attB TNFAIP3(we make this time) is 35ng/uL

2. BP reaction
We adjusted solution (vials) on 1.5mL tube to next composition.
attB TNFAIP3(35ng/μL) 2μL 
pDONR(150ng/μL) 1μL 
TE buffer 5μL 
Total 8μL 

We added BP Clonase Ⅱ enzyme mix-2uL to this vials.
We incubated these vials for 2hour at 25˚C. Next we did BP reaction

3. Transformation
We added BP TNFAIP3-2uL to XL1-Blue-100uL and we did Transformation.
Finally we spread 10uL, 40uL and 100uL on plate of LB Kanamycin(+) and we keep 37℃ overnight.


August 22nd


TNFA and API2

We separated six colonies from 40uL spread plate of BP TNFAIP3 (make August 21 ) .
We cultivated these colonies on 2.5mL Kanamycin(+) LB culture medium and we kept shaking this medium overnight.


August 23rd


TNFA and API2



August 24th


August 25th


August 26th


The female-virgin flies (w; Δ2,3) and male flies (yw) were collected for microinjection. The collected flies were kept in the 25℃ incubator separately.