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Team:KIT-Kyoto/Notebook-week3
From 2012.igem.org
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<strong>Parts</strong> | <strong>Parts</strong> | ||
| + | <br><br> | ||
| + | <Table Border Cellspacing="0"> | ||
| + | <Tr><Td>All DNA</Td><Td>500ng</Td><Td>1ug</Td></Tr> | ||
| + | <tr><td>pEGFP-C</td><td>3uL</td><Td>6uL</Td></tr> | ||
| + | <Tr><Td>H buffer(TOYOBO)</Td><Td>5uL</Td><Td>5uL</Td></Tr> | ||
| + | <tr><td>BamHⅠ(TOYOBO)</td><td>1uL</td><Td>1uL</Td></tr><tr> | ||
| + | <td>BglⅡ(TOYOBO)</td><td>1uL</td><Td>1uL</Td></tr><tr> | ||
| + | <td>100×BSA</td><td>0.5uL</td><Td>0.5uL</Td></tr><tr> | ||
| + | <td>dH<sub>2</sub>O</td><td>39.5uL</td><Td>36.5uL</Td></tr><tr> | ||
| + | <td>Total</td><td>50uL</td><Td>50uL</Td></tr> | ||
| + | </Table> | ||
<br><br> | <br><br> | ||
<img src="https://static.igem.org/mediawiki/2012/d/d0/0825kit.png" width="500" height="300"> | <img src="https://static.igem.org/mediawiki/2012/d/d0/0825kit.png" width="500" height="300"> | ||
<br><BR> | <br><BR> | ||
| + | <Table Border Cellspacing="0"> | ||
| + | <tr><td>DNA sample</td><td>23uL</td></tr> | ||
| + | <Tr><Td>4 buffer(NEB)</Td><Td>5uL</Td></Tr> | ||
| + | <tr><td>EcoRⅠ-HF(NEB)</td><td>1uL</td></tr><tr> | ||
| + | <td>SpeⅠ(NEB)</td><td>1.5uL</td></tr> | ||
| + | <td>100×BSA</td><td>0.5uL</td></tr> | ||
| + | <td>dH<sub>2</sub>O</td><td>19uL</td></tr> | ||
| + | <td>Total<td>50uL</td></tr> | ||
| + | </Table> | ||
| + | <br><BR> | ||
| + | <Table Border Cellspacing="0"> | ||
| + | <Tr><Td></Td><Td>All samples</Td></Tr> | ||
| + | <tr><td>DNA template</td><td>0.5uL</td></tr> | ||
| + | <Tr><Td>10× KOD plus buffer</Td><Td>10uL</Td></Tr> | ||
| + | <tr><td>2mM dNTPs</td><td>10uL</td></tr><tr> | ||
| + | <td>25mM MgSO4</td><td>3.2uL</td></tr> | ||
| + | <td>10P 5’ primer</td><td>3uL</td></tr> | ||
| + | <td>10P 3’ primer</td><td>3uL</td></tr> | ||
| + | <td>KOD plus</td><td>2uL</td></tr> | ||
| + | <td>dH<sub>2</sub>O</td><td>68.3uL</td></tr> | ||
| + | <td>Total<td>100uL</td></tr> | ||
| + | </Table> | ||
| + | <br><BR> | ||
| + | <Table Border Cellspacing="0"> | ||
| + | <Tr><Td>Tempreture</Td><Td>Time</Td><Td>Circle</Td></Tr> | ||
| + | <tr><td>95℃</td><td>2min</td><Td></Td></tr> | ||
| + | <Tr><Td>95℃</Td><Td>15sec</Td><Td>25 cycle</Td></Tr> | ||
| + | <tr><td>58℃</td><td>30min(UAS) or 2min10sec(pSB1C3)</td><Td>25 cycle</Td></tr><tr> | ||
| + | <td>68℃</td><td>2min30sec</td><Td>25 cycle</Td></tr> | ||
| + | <td>68℃</td><td>2min30sec</td><Td></Td></tr> | ||
| + | <td>14℃</td><td>∞</td><Td></Td></tr> | ||
| + | </Table> | ||
| + | <br><br> | ||
<h2>August 26th</h2> | <h2>August 26th</h2> | ||
<br> | <br> | ||
Revision as of 11:25, 23 September 2012
August 20thTNFA 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 21stTNFA 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 22ndTNFA 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 23rdTNFA and API2 
Parts August 24thTNFA and API2 Parts August 25thTNFA and API2 Parts
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. Parts 
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