Team:KIT-Kyoto/Notebook-week3

From 2012.igem.org

(Difference between revisions)
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<h2>August 20th</h2>
<h2>August 20th</h2>
<br>
<br>
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<strong>*TNFA and API2</strong>
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<br><br>
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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>
<br>
<br>
-
<strong>*TNFA and API2</strong>
+
 
-
<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>
<br>
<br>
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<strong>*TNFA and API2</strong>
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<br><br>
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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>
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<strong>*TNFA and API2</strong>
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<br><br>
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<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>
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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>
+
 
-
<br><br>
+
-
<strong>*Parts</strong>
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-
<br><br>
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<h2>August 25th</h2>
<h2>August 25th</h2>
<br>
<br>
-
<strong>*TNFA and API2</strong>
+
 
-
<br><br>
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-
<strong>*Parts</strong>
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<br><br>
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<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
<Tr><Td> All DNA </Td><Td> 500ng </Td><Td> 1ug </Td></Tr>
<Tr><Td> All DNA </Td><Td> 500ng </Td><Td> 1ug </Td></Tr>
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<h2>August 26th</h2>
<h2>August 26th</h2>
<br>
<br>
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<strong>*TNFA and API2</strong>
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<br><br>
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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>
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<strong>*Parts</strong>
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<br><br>
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<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">
<br>
<br>

Revision as of 02:47, 25 September 2012






August 20th


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


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


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




August 24th


August 25th


 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


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.