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Team:KIT-Kyoto/Notebook-week3
From 2012.igem.org
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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> | |
<strong>Composition</strong> | <strong>Composition</strong> | ||
<Table Border Cellspacing="0"> | <Table Border Cellspacing="0"> | ||
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| - | We separated 54uL×2 from 108uL (sample) and we did electrophoresis (54uL sample and 1kb marker 5uL).We separated gel containing DNA.<br><br> | + | We separated 54uL×2 from 108uL (sample) and we did electrophoresis (54uL sample and 1kb marker 5uL). |
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| + | We separated gel containing DNA.<br><br> | ||
<strong>Results</strong><br><br> | <strong>Results</strong><br><br> | ||
<img src="https://static.igem.org/mediawiki/2012/b/b8/0820.png" width="500" height="300"> | <img src="https://static.igem.org/mediawiki/2012/b/b8/0820.png" width="500" height="300"> | ||
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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> | |
<strong>Results</strong> | <strong>Results</strong> | ||
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2. BP reaction<br> | 2. BP reaction<br> | ||
| - | + | We adjusted solution (vials) on 1.5mL tube to next composition.<br> | |
<Table Border Cellspacing="0"> | <Table Border Cellspacing="0"> | ||
<Tr><Td> attB TNFAIP3(35ng/μL)</Td><Td> 2μL </Td></Tr> | <Tr><Td> attB TNFAIP3(35ng/μL)</Td><Td> 2μL </Td></Tr> | ||
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| - | + | We added BP Clonase Ⅱ enzyme mix-2uL to this vials.<br> | |
| - | + | We incubated these vials for 2hour at 25˚C. Next we did BP reaction<br><br> | |
3. Transformation<br> | 3. Transformation<br> | ||
| - | + | We added BP TNFAIP3-2uL to XL1-Blue-100uL and we did Transformation.<br> | |
| - | + | Finally we spread 10uL, 40uL and 100uL on plate of LB Kanamycin(＋) and we keep 37℃ overnight.<br> | |
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Revision as of 09:18, 24 September 2012
August 20th*TNFA and API2 1. Reproduction of DNA from gel We did agarose gel electrophoresis (0.7% gel). Composition
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.
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
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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