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Team:KIT-Kyoto/c6h12o6
From 2012.igem.org
(Difference between revisions)
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<Table Border Cellspacing="0"> | <Table Border Cellspacing="0"> | ||
| - | <Tr><Td> | + | <Tr><Td></Td><Td>sample1</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>10ng/μL TNFAIP3</Td><Td>6μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>10× KOD plus buffer</Td><Td>10μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>2mM dNTPs</Td><Td>10μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>25mM MgSO<sub>4</sub></Td><Td>3.2μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>10P 5'primer</Td><Td>3μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>10P 3'primer</Td><Td>3μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>KOD plus</Td><Td>2μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>dH<sub>2</sub>O</Td><Td>62.8μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>total</Td><Td>100μL</Td></Tr> |
</Table> | </Table> | ||
<br> | <br> | ||
| Line 41: | Line 41: | ||
<Table Border Cellspacing="0"> | <Table Border Cellspacing="0"> | ||
| - | <Tr><Td> | + | <Tr><Td></Td><Td>sample1</Td><Td>sample2</Td><Td>sample3</Td><Td>sample4</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>10ng/μL API2-MALT1</Td><Td>2μL</Td><Td>2μL</Td><Td>1μL</Td><Td>1μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>10× KOD plus buffer</Td><Td>2μL</Td><Td>2μL</Td><Td>2μL</Td><Td>2μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>2mM dNTPs</Td><Td>2μL</Td><Td>2μL</Td><Td>2μL</Td><Td>2μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>25mM MgSO<sub>4</sub></Td><Td>0.8μL</Td><Td>0.8μL</Td><Td>0.8μL</Td><Td>0.8μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>10P 5'primer</Td><Td>0.6μL</Td><Td>0.6μL</Td><Td>0.6μL</Td><Td>0.6μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>10P 3'primer</Td><Td>0.6μL</Td><Td>0.6μL</Td><Td>0.6μL</Td><Td>0.6μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>KOD plus</Td><Td>0.4μL</Td><Td>0.4μL</Td><Td>0.4μL</Td><Td>0.4μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>dH<sub>2</sub>O</Td><Td>11.6μL</Td><Td>11.6μL</Td><Td>12.6μL</Td><Td>12.6μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>total</Td><Td>20μL</Td><Td>20μL</Td><Td>20μL</Td><Td>20μL</Td></Tr> |
</Table> | </Table> | ||
<br> | <br> | ||
| Line 84: | Line 84: | ||
<Table Border Cellspacing="0"> | <Table Border Cellspacing="0"> | ||
| - | <Tr><Td> | + | <Tr><Td></Td><Td>1-2</Td><Td>1-3</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>DNA sample</Td><Td>10μL</Td><Td>20μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>6×Dye</Td><Td>2μL</Td><Td>4μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>total</Td><Td>12μL</Td><Td>25μL</Td></Tr> |
</Table> | </Table> | ||
<br> | <br> | ||
| Line 95: | Line 95: | ||
<Tr><Td>DNA sample</Td><Td>90μL</Td></Tr> | <Tr><Td>DNA sample</Td><Td>90μL</Td></Tr> | ||
<Tr><Td>6×Dye</Td><Td>18μL</Td></Tr> | <Tr><Td>6×Dye</Td><Td>18μL</Td></Tr> | ||
| - | <Tr><Td> | + | <Tr><Td>total</Td><Td>108μL</Td></Tr> |
</Table> | </Table> | ||
<br> | <br> | ||
<Table Border Cellspacing="0"> | <Table Border Cellspacing="0"> | ||
| - | <Tr><Td> | + | <Tr><Td>10ng/μL API2-MALT1</Td><Td>5μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>10×KOD plus buffer</Td><Td>10μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>2mM dNTPs</Td><Td>10μL</Td></Tr> |
| - | <Tr><Td>4 | + | <Tr><Td>25mM MgSO<sub>4</sub></Td><Td>4μL</Td></Tr> |
| - | <Tr><Td>5 | + | <Tr><Td>10P 5'primer</Td><Td>3μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>10P 3'primer</Td><Td>3μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>KOD plus</Td><Td>2μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>dH<sub>2</sub>O</Td><Td>63μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>total</Td><Td>100μL</Td></Tr> |
</Table> | </Table> | ||
<br> | <br> | ||
| Line 145: | Line 145: | ||
<Table Border Cellspacing="0"> | <Table Border Cellspacing="0"> | ||
| - | <Tr><Td> | + | <Tr><Td>attB TNFAIP3(80ng/μL)</Td><Td>1μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>pDONR(455ng/μL)</Td><Td>0.35μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>TE buffer</Td><Td>7.65</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>total</Td><Td>9μL</Td></Tr> |
</Table> | </Table> | ||
<br> | <br> | ||
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<Table Border Cellspacing="0"> | <Table Border Cellspacing="0"> | ||
| - | <Tr><Td> | + | <Tr><Td></Td><Td>sample1</Td><Td>sample2</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>10ng/μL AIP2-MALT1</Td><Td>2.5μL</Td><Td>2.5μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>10×KOD plus buffer</Td><Td>5μL</Td><Td>5μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>2mM dNTPs</Td><Td>5μL</Td><Td>5μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>25mM MgSO<sub>4</sub></Td><Td>2μL</Td><Td>2.4μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>10P 5'primer</Td><Td>1.5μL</Td><Td>1.5μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>10P 3'primer</Td><Td>1.5μL</Td><Td>1.5μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>KOD plus</Td><Td>1μL</Td><Td>1μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>dH<sub>2</sub></Td><Td>31.5μL</Td><Td>31.1μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>total</Td><Td>50μL</Td><Td>50μL</Td></Tr> |
</Table> | </Table> | ||
<br> | <br> | ||
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<Tr><Td></Td><Td>pDONR</Td><Td>BP TNFA-1</Td><Td>BP TNFA-2</Td></Tr> | <Tr><Td></Td><Td>pDONR</Td><Td>BP TNFA-1</Td><Td>BP TNFA-2</Td></Tr> | ||
<Tr><Td>DNA sample</Td><Td>1μL</Td><Td>1μL</Td><Td>1μL</Td></Tr> | <Tr><Td>DNA sample</Td><Td>1μL</Td><Td>1μL</Td><Td>1μL</Td></Tr> | ||
| - | <Tr><Td> | + | <Tr><Td>6×Dye</Td><Td>1μL</Td><Td>1μL</Td><Td>1μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>dH<sub>2</sub>O</Td><Td>4μL</Td><Td>4μL</Td><Td>4μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>total</Td><Td>6μL</Td><Td>6μL</Td><Td>6μL</Td></Tr> |
</Table> | </Table> | ||
<br> | <br> | ||
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<Table Border Cellspacing="0"> | <Table Border Cellspacing="0"> | ||
| - | <Tr><Td> | + | <Tr><Td>BP TNFA-2</Td><Td>2μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>pTFW(Destination vector)</Td><Td>0.5μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>TE buffer</Td><Td>6.5μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>total</Td><Td>9μL</Td></Tr> |
</Table> | </Table> | ||
<br> | <br> | ||
| Line 303: | Line 303: | ||
<Table Border Cellspacing="0"> | <Table Border Cellspacing="0"> | ||
| - | <Tr><Td> | + | <Tr><Td>DNA sample</Td><Td>1μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>10×H buffer</Td><Td>0.5μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>EcoRⅠ</Td><Td>0.2μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>dH<sub>2</sub>O</Td><Td>3.3μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>total</Td><Td>5μL</Td></Tr> |
</Table> | </Table> | ||
<br> | <br> | ||
| Line 313: | Line 313: | ||
<Table Border Cellspacing="0"> | <Table Border Cellspacing="0"> | ||
| - | <Tr><Td> | + | <Tr><Td></Td><Td>1-2</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>50ng/μL LR TNFA-1</Td><Td>1μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>10×rTaq buffer</Td><Td>2μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>2mM dNTPs</Td><Td>2μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>25mM MgCl<sub>2</sub></Td><Td>0.8μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>10P 5'primer</Td><Td>0.6μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>10P 3'primer</Td><Td>0.6μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>rTaq</Td><Td>0.4μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>dH<sub>2</sub>O</Td><Td>12.6μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>total</Td><Td>20μL</Td></Tr> |
</Table> | </Table> | ||
<br> | <br> | ||
| Line 411: | Line 411: | ||
<Table Border Cellspacing="0"> | <Table Border Cellspacing="0"> | ||
<Tr><Td>1-1</Td><Td>1μL</Td></Tr> | <Tr><Td>1-1</Td><Td>1μL</Td></Tr> | ||
| - | <Tr><Td> | + | <Tr><Td>10×M buffer</Td><Td>0.5μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>Hind Ⅲ</Td><Td>0.2μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>dH<sub>2</sub>O</Td><Td>3.3μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>total</Td><Td>5μL</Td></Tr> |
</Table> | </Table> | ||
<br> | <br> | ||
| Line 438: | Line 438: | ||
<Table Border Cellspacing="0"> | <Table Border Cellspacing="0"> | ||
<Tr><Td>1-1</Td><Td>sample1</Td></Tr> | <Tr><Td>1-1</Td><Td>sample1</Td></Tr> | ||
| - | <Tr><Td> | + | <Tr><Td>10ng/μL TNFAIP3</Td><Td>6μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>10×KOD plus buffer</Td><Td>10μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>2mM dNTPs</Td><Td>10μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>25mM MgSO<sub>4</sub></Td><Td>3.2μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>10P 5'primer</Td><Td>3μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>10P 3'primer</Td><Td>3μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>KOD plus</Td><Td>2μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>dH<sub>2</sub>O</Td><Td>62.8μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>total</Td><Td>100μL</Td></Tr> |
</Table> | </Table> | ||
<br> | <br> | ||
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<Table Border Cellspacing="0"> | <Table Border Cellspacing="0"> | ||
| - | <Tr><Td> | + | <Tr><Td></Td><Td>1-2</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>DNA sample</Td><Td>90μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>6×Dye</Td><Td>18μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>total</Td><Td>108μL</Td></Tr> |
</Table> | </Table> | ||
<br> | <br> | ||
| Line 597: | Line 597: | ||
<Table Border Cellspacing="0"> | <Table Border Cellspacing="0"> | ||
| - | <Tr><Td> | + | <Tr><Td>attB TNFAIP3(35ng/μL)</Td><Td>2μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>pDONR(150ng/μL)</Td><Td>1μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>TE buffer</Td><Td>5μL</Td></Tr> |
| - | <Tr><Td> | + | <Tr><Td>total</Td><Td>8μL</Td></Tr> |
</Table> | </Table> | ||
<br> | <br> | ||
Revision as of 01:04, 9 September 2012
1 August
| sample1 | |
| 10ng/μL TNFAIP3 | 6μL |
| 10× KOD plus buffer | 10μL |
| 2mM dNTPs | 10μL |
| 25mM MgSO4 | 3.2μL |
| 10P 5'primer | 3μL |
| 10P 3'primer | 3μL |
| KOD plus | 2μL |
| dH2O | 62.8μL |
| total | 100μL |
| temperature | time | cycle |
| 95°C | 2min | |
| 95°C | 15sec | 25cycle |
| 60°C | 30sec | 25cycle |
| 68°C | 2min30sec | 25cycle |
| 68°C | 2min30sec | |
| 14°C | ∞ |
| sample1 | sample2 | sample3 | sample4 | |
| 10ng/μL API2-MALT1 | 2μL | 2μL | 1μL | 1μL |
| 10× KOD plus buffer | 2μL | 2μL | 2μL | 2μL |
| 2mM dNTPs | 2μL | 2μL | 2μL | 2μL |
| 25mM MgSO4 | 0.8μL | 0.8μL | 0.8μL | 0.8μL |
| 10P 5'primer | 0.6μL | 0.6μL | 0.6μL | 0.6μL |
| 10P 3'primer | 0.6μL | 0.6μL | 0.6μL | 0.6μL |
| KOD plus | 0.4μL | 0.4μL | 0.4μL | 0.4μL |
| dH2O | 11.6μL | 11.6μL | 12.6μL | 12.6μL |
| total | 20μL | 20μL | 20μL | 20μL |
| temperature | time | cycle |
| 95°C | 2min | |
| 95°C | 15sec | 25cycle |
| 50°C(sample1,3)or55°C(sample2,4) | 30sec | 25cycle |
| 68°C | 3min30sec | 25cycle |
| 68°C | 3min30sec | |
| 14°C | ∞ |
2 August
| 1-2 | 1-3 | |
| DNA sample | 10μL | 20μL |
| 6×Dye | 2μL | 4μL |
| total | 12μL | 25μL |
| 1-2 | |
| DNA sample | 90μL |
| 6×Dye | 18μL |
| total | 108μL |
| 10ng/μL API2-MALT1 | 5μL |
| 10×KOD plus buffer | 10μL |
| 2mM dNTPs | 10μL |
| 25mM MgSO4 | 4μL |
| 10P 5'primer | 3μL |
| 10P 3'primer | 3μL |
| KOD plus | 2μL |
| dH2O | 63μL |
| total | 100μL |
| temperature | time | cycle |
| 95°C | 2min | |
| 95°C | 15sec | 35cycle |
| 55°C | 30sec | 35cycle |
| 68°C | 3min30sec | 35cycle |
| 68°C | 3min30sec | |
| 14°C | ∞ |
Result
3 August
| attB TNFAIP3(80ng/μL) | 1μL |
| pDONR(455ng/μL) | 0.35μL |
| TE buffer | 7.65 |
| total | 9μL |
| sample1 | sample2 | |
| 10ng/μL AIP2-MALT1 | 2.5μL | 2.5μL |
| 10×KOD plus buffer | 5μL | 5μL |
| 2mM dNTPs | 5μL | 5μL |
| 25mM MgSO4 | 2μL | 2.4μL |
| 10P 5'primer | 1.5μL | 1.5μL |
| 10P 3'primer | 1.5μL | 1.5μL |
| KOD plus | 1μL | 1μL |
| dH2 | 31.5μL | 31.1μL |
| total | 50μL | 50μL |
| temperature | time | cycle |
| 95°C | 2min | |
| 95°C | 15sec | 35cycle |
| 55°C | 30sec | 35cycle |
| 68°C | 3min30sec | 35cycle |
| 68°C | 3min30sec | |
| 14°C | ∞ |
4 August
5 August
6 August
7 August
| pDONR | BP TNFA-1 | BP TNFA-2 | |
| DNA sample | 1μL | 1μL | 1μL |
| 6×Dye | 1μL | 1μL | 1μL |
| dH2O | 4μL | 4μL | 4μL |
| total | 6μL | 6μL | 6μL |
| BP TNFA-2 | 2μL |
| pTFW(Destination vector) | 0.5μL |
| TE buffer | 6.5μL |
| total | 9μL |
8 August
9 Augus
| DNA sample | 1μL |
| 10×H buffer | 0.5μL |
| EcoRⅠ | 0.2μL |
| dH2O | 3.3μL |
| total | 5μL |
| 1-2 | |
| 50ng/μL LR TNFA-1 | 1μL |
| 10×rTaq buffer | 2μL |
| 2mM dNTPs | 2μL |
| 25mM MgCl2 | 0.8μL |
| 10P 5'primer | 0.6μL |
| 10P 3'primer | 0.6μL |
| rTaq | 0.4μL |
| dH2O | 12.6μL |
| total | 20μL |
| temperature | time | cycle |
| 95°C | 2min | |
| 95°C | 15sec | 25cycle |
| 55°C | 30sec | 25cycle |
| 68°C | 2min30sec | 25cycle |
| 68°C | 2min30sec | |
| 14°C | ∞ |
10 August
11 August
12 August
13 August
| 1-1 | 1μL |
| 10×M buffer | 0.5μL |
| Hind Ⅲ | 0.2μL |
| dH2O | 3.3μL |
| total | 5μL |
14 August
| 1-1 | sample1 |
| 10ng/μL TNFAIP3 | 6μL |
| 10×KOD plus buffer | 10μL |
| 2mM dNTPs | 10μL |
| 25mM MgSO4 | 3.2μL |
| 10P 5'primer | 3μL |
| 10P 3'primer | 3μL |
| KOD plus | 2μL |
| dH2O | 62.8μL |
| total | 100μL |
| temperature | time | cycle |
| 95°C | 2min | |
| 95°C | 15sec | 25cycle |
| 60°C | 30sec | 25cycle |
| 68°C | 2min30sec | 25cycle |
| 68°C | 2min30sec | |
| 14°C | ∞ |
15 August
16 August
17 August
18 August
19 August
20 August
| 1-2 | |
| DNA sample | 90μL |
| 6×Dye | 18μL |
| total | 108μL |
21 August
| attB TNFAIP3(35ng/μL) | 2μL |
| pDONR(150ng/μL) | 1μL |
| TE buffer | 5μL |
| total | 8μL |
