Team:KIT-Kyoto/Notebook-week3

From 2012.igem.org

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1. Reproduction of DNA from gel<br>
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1. Isolation of DNA fragment from the gel
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 We did agarose gel electrophoresis (0.7% gel).<br><br>
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Sample applied to the electrophoresis
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<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).
 
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We separated gel containing DNA.<br><br>
 
<strong>Results</strong><br><br>
<strong>Results</strong><br><br>
<img src="http://2012.igem.org/wiki/images/b/b8/0820.png" width="500" height="300">
<img src="http://2012.igem.org/wiki/images/b/b8/0820.png" width="500" height="300">
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We isolated DNA from these gels by QIA quick Gel Extraction Kit.<br>
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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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Finally we melt DNA to TE Buffer(40uL)<br>
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1. Density measurement of attB TNFAIP3<br>
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1. Measuring the concentration of the attB TNFAIP3 DNA fragment
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 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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The order of sample applied to the electrophoresis
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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,
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<strong>Results</strong>
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<br><br>
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<strong>Result</strong>
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<img src="http://2012.igem.org/wiki/images/6/67/0821kit.png" width="500" height="300">
<img src="http://2012.igem.org/wiki/images/6/67/0821kit.png" width="500" height="300">
<br><br>
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We estimated attB TNFAIP3(we make this time) is 35ng/uL<br><br>
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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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<br><br>
2. BP reaction<br>
2. BP reaction<br>
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 We adjusted solution (vials) on 1.5mL tube to next composition.<br>
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 1.5mL tube<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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<Tr><Td> Total </Td><Td> 8μL </Td></Tr>
<Tr><Td> Total </Td><Td> 8μL </Td></Tr>
</Table>
</Table>
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<br><br>
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We added 2uL of BP Clonase Ⅱ enzyme mix to this solution and incubate it for 2 hour.  
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 We added BP Clonase Ⅱ enzyme mix-2uL to this vials.<br>
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<br><br>
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 We incubated these vials for 2hour at 25˚C. Next we did BP reaction<br><br>
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3. Transformation<br>
3. Transformation<br>
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 We added BP TNFAIP3-2uL to XL1-Blue-100uL and we did Transformation.<br>
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 We added 100uL of XL1-Blue to the BP reaction products to do transformation.
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 Finally we spread 10uL, 40uL and 100uL on plate of LB Kanamycin(+) and we keep 37℃ overnight.<br>
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We separated six colonies from 40uL spread plate of BP TNFAIP3 (make August 21 ) .<br>
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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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We cultivated these colonies on 2.5mL Kanamycin(+) LB culture medium and we kept shaking this medium overnight.<br>
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Revision as of 14:25, 25 September 2012






August 20th


1. Isolation of DNA fragment from the gel Sample applied to the electrophoresis

Composition
 a product of PCR attB TNFAIP3(8/1) 
 DNA sample        90μL
 6×Dye        18μL
 Total        108μL


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 21st


1. 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
 attB TNFAIP3(35ng/μL) 2μL 
 pDONR(150ng/μL)  1μL 
 TE buffer  5μL 
 Total  8μL 


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 22nd


We isolated 6 colonies from a plate and incubated in 2.5mL of Kanamycin(+) LB liquid culture medium for 16 hours.

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.