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Team:KIT-Kyoto/c6h12o6
From 2012.igem.org
August 1st
*TNFAIP3
Since it takes at least one month to establish transgenic flies, we decided to first construct the P-element plasmid for microinjection into Drosophia embryos.
① We performed PCR in the following conditions.
primer
(ⅰ)TNFAIP3
Composition for reaction
| 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 |
Reaction conditions
| temperature | time | cycle |
| 95°C | 2min. | |
| 95°C | 15sec | 25cycle |
| 60°C | 30sec | 25cycle |
| 68°C | 2min30sec | 25cycle |
| 68°C | 2min30sec | |
| 14°C | ∞ |
August 2nd
*TNFA and API2
① PCR product check
We run 0.7% agarose gel electorophoresis in the following conditions. Composition
| 8/1 the attB TNFAIP3 PCR products | |
| DNA sample | 10μL |
| 6×Dye | 2μL |
| total | 12μL |
The order of sample application
Left : 1kb marker(5uL)
Right: attB TNFAIP3
Results
② DNA purification from gel
We electrophoresed in 0.7% agarose gel in the following conditions.
Composition
| 8/1 attB TNFAIP3 PCRproduct | |
| DNA sample | 90μL |
| 6×Dye | 18μL |
| total | 108μL |
We applied half of 108 uL of sample to each of two lanes of Agaroe gel with 5uL of 1kb marker.Then we detected the DNA band in the gel.
Results
We purifed DNA from the gel by QIA Quick Gel Extraction Kit.
・Eatimation of the amount of attB TNFAIP3 DNA fragments by electrophoresis.
Order of samples applied to the agarose gel
The samples were electrophoresed in 0.7% agarose gel in following order.
Right to left:3uL of 1kb marker 1uL of 5 fold dilution of attB TNFAIP3 DNA fragments,1uL of 10 fold dilution of attB TNFAIP3 DNA fragments, 1uL of 20 fold dilution of attB TNFAIP3 DNA fragments, 1uL of 1kb marker.
Results
We have estimated amount of the attB TNFAIP3 DNA to be 80ng/uL.
Result
August 3rd
*TNFA and API2
①BP reaction
BP reaction was carried out in the following conditions.
| attB TNFAIP3(80ng/μL) | 1μL |
| pDONR(455ng/μL) | 0.35μL |
| TE buffer | 7.65 |
| total | 9μL |
One uL of BP Clonase Ⅱ enzyme mix was added to this solution and incubated for 2.5 hours.
② Transformation of E.coli by BP reaction products
2uL of BP TNFA reaction product was added to 100uL of E.coli XL1-Blue to do transform ation.
At last, cells were divided into 10,40,100,and 200 uL,and spreaded on LB Kanamycin(+)plate,Plats wera incubated for 16 hours at 37°C.
August 4th
*TNFA and API2
Four independent 4 colonies were picked up from the plate incubated from 8/3 as described in ②,then cultured in 2.5mL of LB Kanamycin(+) liquid culture medium for 16 hours.
August 5th
*TNFA and API2
Plasmid DNAs were isolated from E.coli cultured in 4 of LB liquid culture medium which we made on 8/4 by the alkaline-lysis method, we added the DNA to 20uL TE and added 10uL of the solution to 2uL of 6×Dye. Then we electrophoresed it with marker, 2uL of pDONR+ 2uL of 6×Dye+ 8uL of TE,in 0.7% agarose gel in following order (from left to right)
Left control pDONR DNA, BP TNFA-1,-2,-3,-4
Results
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.
