Team:KIT-Kyoto/Notebook-week3

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<h2>August 23rd</h2>
 
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<h2>August 24th</h2>
 
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<h2>August 25th</h2>
 
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<Tr><Td>　All DNA　</Td><Td>　500ng　</Td><Td>　1ug　</Td></Tr>
 
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<tr><td>　pEGFP-C　</td><td>　3uL　</td><Td>　6uL　</Td></tr>
 
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<Tr><Td>　H buffer(TOYOBO)　</Td><Td>　5uL　</Td><Td>　5uL　</Td></Tr>
 
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<tr><td>　BamHⅠ(TOYOBO)　</td><td>　1uL　</td><Td>　1uL　</Td></tr><tr>
 
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<td>　BglⅡ(TOYOBO)　</td><td>　1uL　</td><Td>　1uL　</Td></tr><tr>
 
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<td>　100×BSA　</td><td>　0.5uL　</td><Td>　0.5uL　</Td></tr><tr>
 
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<td>　dH<sub>2</sub>O　</td><td>　39.5uL　</td><Td>　36.5uL　</Td></tr><tr>
 
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<td>　Total　</td><td>　50uL　</td><Td>　50uL　</Td></tr>
 
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<tr><td>　DNA sample　</td><td>　23uL　</td></tr>
 
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<Tr><Td>　4 buffer(NEB)　</Td><Td>　5uL　</Td></Tr>
 
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<tr><td>　EcoRⅠ-HF(NEB)　</td><td>　1uL　</td></tr><tr>
 
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<td>　SpeⅠ(NEB)　</td><td>　1.5uL　</td></tr>
 
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<td>　100×BSA　</td><td>　0.5uL　</td></tr>
 
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<td>　dH<sub>2</sub>O　</td><td>　19uL　</td></tr>
 
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<td>　Total　<td>　50uL　</td></tr>
 
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<Tr><Td></Td><Td>　All samples　</Td></Tr>
 
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<tr><td>　DNA template　</td><td>　0.5uL　</td></tr>
 
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<Tr><Td>　10× KOD plus buffer　</Td><Td>　10uL　</Td></Tr>
 
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<tr><td>　2mM dNTPs　</td><td>　10uL　</td></tr><tr>
 
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<td>　25mM MgSO4　</td><td>　3.2uL　</td></tr>
 
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<td>　10P 5’ primer　</td><td>　3uL　</td></tr>
 
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<td>　10P 3’ primer　</td><td>　3uL　</td></tr>
 
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<td>　KOD plus　</td><td>　2uL　</td></tr>
 
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<td>　dH<sub>2</sub>O　</td><td>　68.3uL　</td></tr>
 
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<td>　Total　<td>　100uL　</td></tr>
 
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<Tr><Td>　Temperature　</Td><Td>　Time　</Td><Td>　Circle　</Td></Tr>
 
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<tr><td>　　95℃</td><td>　2min</td><Td></Td></tr>
 
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<Tr><Td>　　95℃</Td><Td>　15sec</Td><Td>　25 cycle　</Td></Tr>
 
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<tr><td>　　58℃</td><td>　30min(UAS) or 2min10sec(pSB1C3)</td><Td>　25 cycle　</Td></tr><tr>
 
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<td>　　68℃</td><td>　2min30sec</td><Td>　25 cycle　</Td></tr>
 
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<td>　　68℃</td><td>　2min30sec</td><Td></Td></tr>
 
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<td>　　14℃</td><td>　∞</td><Td></Td></tr>
 
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<h2>August 26th</h2>
<h2>August 26th</h2>
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<div align="right"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week4">>>>>>>>>>WEEK4</a></div>
<div align="right"><a href="https://2012.igem.org/Team:KIT-Kyoto/Notebook-week4">>>>>>>>>>WEEK4</a></div>
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Revision as of 12:14, 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 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.

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