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Team:KIT-Kyoto/c6h12o6
From 2012.igem.org
August 1st
*TNFA and API2
| 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 | ∞ |
August 2nd
*TNFA and API2
| 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
August 3rd
*TNFA and API2
| 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 | ∞ |
August 4th
*TNFA and API2
August 5th
*TNFA and API2
August 6th
*TNFA and API2
August 7th
*TNFA and API2
| 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 |
WEEK１終わり
August 8th
*TNFA and API2
August 9th
*TNFA and API2
| 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 | ∞ |
August 10th
*TNFA and API2
August 13th
*TNFA and API2
| 1-1 | 1μL |
| 10×M buffer | 0.5μL |
| Hind Ⅲ | 0.2μL |
| dH2O | 3.3μL |
| total | 5μL |
August 14th
*TNFA and API2
| 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 | ∞ |
WEEK2終わり
August 20th
TNFA and API2
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
TNFA 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.
| 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
TNFA 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.
