Team:KIT-Kyoto/Notebook-week3
From 2012.igem.org
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- | 1. | + | 1. Isolation of DNA fragment from the gel |
- | + | Sample applied to the electrophoresis | |
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<strong>Composition</strong> | <strong>Composition</strong> | ||
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<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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- | + | DNA was isolated from the agarose gel by QIA Quick Gel Extraction Kit, then the DNA was dissolved in 40 uL of TE. | |
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- | 1. | + | 1. Measuring the concentration of the attB TNFAIP3 DNA fragment |
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+ | The order of sample applied to the electrophoresis | ||
+ | <br> | ||
+ | 1kbmarker3uL,1uL of 5 fold dilution of attB TNFAIP3, 1uL of 10 fold dilution of attB TNFAIP3 DNA fragment,2uL of 1kbmarker, 1uL of 15 fold dilution of attB TNFAIP3 DNA fragment, 1uL of 20 fold dilution of attB TNFAIP3 DNA fragment, 1kbmarker1uL, | ||
- | <strong> | + | <br><br> |
+ | <strong>Result</strong> | ||
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<img src="https://static.igem.org/mediawiki/2012/6/67/0821kit.png" width="500" height="300"> | <img src="https://static.igem.org/mediawiki/2012/6/67/0821kit.png" width="500" height="300"> | ||
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- | + | The concentration of the isolated attB TNFAIP3 DNA fragments was estimated to be 35ng/uL.<br><br> | |
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2. BP reaction<br> | 2. BP reaction<br> | ||
- | + | 1.5mL tube<br> | |
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<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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<Tr><Td> Total </Td><Td> 8μL </Td></Tr> | <Tr><Td> Total </Td><Td> 8μL </Td></Tr> | ||
</Table> | </Table> | ||
- | <br> | + | <br><br> |
- | + | We added 2uL of BP Clonase Ⅱ enzyme mix to this solution and incubate it for 2 hour. | |
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3. Transformation<br> | 3. Transformation<br> | ||
- | We added | + | We added 100uL of XL1-Blue to the BP reaction products to do transformation. |
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- | We | + | We isolated 6 colonies from a plate and incubated in 2.5mL of Kanamycin(+) LB liquid culture medium for 16 hours. |
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Revision as of 14:25, 25 September 2012
August 20th1. Isolation of DNA fragment from the gel Sample applied to the electrophoresis Composition
Results DNA was isolated from the agarose gel by QIA Quick Gel Extraction Kit, then the DNA was dissolved in 40 uL of TE. August 21st1. Measuring the concentration of the attB TNFAIP3 DNA fragment The order of sample applied to the electrophoresis 1kbmarker3uL,1uL of 5 fold dilution of attB TNFAIP3, 1uL of 10 fold dilution of attB TNFAIP3 DNA fragment,2uL of 1kbmarker, 1uL of 15 fold dilution of attB TNFAIP3 DNA fragment, 1uL of 20 fold dilution of attB TNFAIP3 DNA fragment, 1kbmarker1uL, Result The concentration of the isolated attB TNFAIP3 DNA fragments was estimated to be 35ng/uL. 2. BP reaction 1.5mL tube
We added 2uL of BP Clonase Ⅱ enzyme mix to this solution and incubate it for 2 hour. 3. Transformation We added 100uL of XL1-Blue to the BP reaction products to do transformation. August 22ndWe isolated 6 colonies from a plate and incubated in 2.5mL of Kanamycin(+) LB liquid culture medium for 16 hours. August 26thThe female-virgin flies (w; Δ2,3) and male flies (yw) were collected for microinjection. The collected flies were kept in the 25℃ incubator separately. |