Team:KIT-Kyoto/Notebook-week3

From 2012.igem.org

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<h2>August 20th</h2>
<h2>August 20th</h2>
<br>
<br>
-
<strong>TNFA and API2</strong>
+
<strong>*TNFA and API2</strong>
<br><br>
<br><br>
1. Reproduction of DNA from gel<br>
1. Reproduction of DNA from gel<br>
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<br>
<br>
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<h2>August 21st</h2>
<h2>August 21st</h2>
<br>
<br>
-
<strong>TNFA and API2</strong>
+
<strong>*TNFA and API2</strong>
<br><br>
<br><br>
1. Density measurement of attB TNFAIP3<br>
1. Density measurement of attB TNFAIP3<br>
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<h2>August 22nd</h2>
<h2>August 22nd</h2>
<br>
<br>
-
<strong>TNFA and API2</strong>
+
<strong>*TNFA and API2</strong>
<br><br>
<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>
<br><br>
<br><br>
 +
<h2>August 23rd</h2>
<h2>August 23rd</h2>
<br>
<br>
-
<strong>TNFA and API2</strong>
+
<strong>*TNFA and API2</strong>
<br><br>
<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">
<br>
<br>
<br>
<br>
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<strong>Parts</strong>
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<strong>*Parts</strong>
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<br><br>
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<h2>August 24th</h2>
<h2>August 24th</h2>
<br>
<br>
-
<strong>TNFA and API2</strong>
+
<strong>*TNFA and API2</strong>
<br><br>
<br><br>
-
<strong>Parts</strong>
+
<strong>*Parts</strong>
<br><br>
<br><br>
 +
<h2>August 25th</h2>
<h2>August 25th</h2>
<br>
<br>
-
<strong>TNFA and API2</strong>
+
<strong>*TNFA and API2</strong>
<br><br>
<br><br>
-
<strong>Parts</strong>
+
<strong>*Parts</strong>
<br><br>
<br><br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
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</Table>
</Table>
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<h2>August 26th</h2>
<h2>August 26th</h2>
<br>
<br>
 +
<strong>*TNFA and API2</strong>
 +
<br><br>
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.
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.
<br><br>
<br><br>
-
<strong>Parts</strong>
+
<strong>*Parts</strong>
<br><br>
<br><br>
<img src="http://2012.igem.org/wiki/images/6/6d/0826.png" width="500" height="300">
<img src="http://2012.igem.org/wiki/images/6/6d/0826.png" width="500" height="300">

Revision as of 09:11, 24 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 sample　　　　　　　　90μL
　6×Dye　　　　　　　　18μL
　Total　　　　　　　　108μL

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



*Parts

August 24th


*TNFA and API2

*Parts

August 25th


*TNFA and API2

*Parts

　All DNA　　500ng　　1ug　
　pEGFP-C　　3uL　　6uL　
　H buffer(TOYOBO)　　5uL　　5uL　
　BamHⅠ(TOYOBO)　　1uL　　1uL　
　BglⅡ(TOYOBO)　　1uL　　1uL　
　100×BSA　　0.5uL　　0.5uL　
　dH2O　　39.5uL　　36.5uL　
　Total　　50uL　　50uL　




　DNA sample　　23uL　
　4 buffer(NEB)　　5uL　
　EcoRⅠ-HF(NEB)　　1uL　
　SpeⅠ(NEB)　　1.5uL　
　100×BSA　　0.5uL　
　dH2O　　19uL　
　Total　　50uL　


　All samples　
　DNA template　　0.5uL　
　10× KOD plus buffer　　10uL　
　2mM dNTPs　　10uL　
　25mM MgSO4　　3.2uL　
　10P 5’ primer　　3uL　
　10P 3’ primer　　3uL　
　KOD plus　　2uL　
　dH2O　　68.3uL　
　Total　　100uL　


　Temperature　　Time　　Circle　
　　95℃　2min
　　95℃　15sec　25 cycle　
　　58℃　30min(UAS) or 2min10sec(pSB1C3)　25 cycle　
　　68℃　2min30sec　25 cycle　
　　68℃　2min30sec
　　14℃　∞


August 26th


*TNFA and API2

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.

*Parts


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