Team:KIT-Kyoto/c6h12o6

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1 August

sample1
10ng/μL TNFAIP36μL
10× KOD plus buffer10μL
2mM dNTPs10μL
25mM MgSO43.2μL
10P 5'primer3μL
10P 3'primer3μL
KOD plus2μL
dH2O62.8μL
total100μL

temperaturetimecycle
95°C2min
95°C15sec25cycle
60°C30sec25cycle
68°C2min30sec25cycle
68°C2min30sec
14°C∞

sample1sample2sample3sample4
10ng/μL API2-MALT12μL2μL1μL1μL
10× KOD plus buffer2μL2μL2μL2μL
2mM dNTPs2μL2μL2μL2μL
25mM MgSO40.8μL0.8μL0.8μL0.8μL
10P 5'primer0.6μL0.6μL0.6μL0.6μL
10P 3'primer0.6μL0.6μL0.6μL0.6μL
KOD plus0.4μL0.4μL0.4μL0.4μL
dH2O11.6μL11.6μL12.6μL12.6μL
total20μL20μL20μL20μL

temperaturetimecycle
95°C2min
95°C15sec25cycle
50°C(sample1,3)or55°C(sample2,4)30sec25cycle
68°C3min30sec25cycle
68°C3min30sec
14°C∞

2 August

1-21-3
DNA sample10μL20μL
6×Dye2μL4μL
total12μL25μL

1-2
DNA sample90μL
6×Dye18μL
total108μL

10ng/μL API2-MALT15μL
10×KOD plus buffer10μL
2mM dNTPs10μL
25mM MgSO44μL
10P 5'primer3μL
10P 3'primer3μL
KOD plus2μL
dH2O63μL
total100μL

temperaturetimecycle
95°C2min
95°C15sec35cycle
55°C30sec35cycle
68°C3min30sec35cycle
68°C3min30sec
14°C∞

Result

3 August

attB TNFAIP3(80ng/μL)1μL
pDONR(455ng/μL)0.35μL
TE buffer7.65
total9μL

sample1sample2
10ng/μL AIP2-MALT12.5μL2.5μL
10×KOD plus buffer5μL5μL
2mM dNTPs5μL5μL
25mM MgSO42μL2.4μL
10P 5'primer1.5μL1.5μL
10P 3'primer1.5μL1.5μL
KOD plus1μL1μL
dH231.5μL31.1μL
total50μL50μL

temperaturetimecycle
95°C2min
95°C15sec35cycle
55°C30sec35cycle
68°C3min30sec35cycle
68°C3min30sec
14°C∞

4 August

5 August

6 August

7 August

pDONRBP TNFA-1BP TNFA-2
DNA sample1μL1μL1μL
6×Dye1μL1μL1μL
dH2O4μL4μL4μL
total6μL6μL6μL

BP TNFA-22μL
pTFW(Destination vector)0.5μL
TE buffer6.5μL
total9μL

8 August

9 Augus

DNA sample1μL
10×H buffer0.5μL
EcoRⅠ0.2μL
dH2O3.3μL
total5μL

1-2
50ng/μL LR TNFA-11μL
10×rTaq buffer2μL
2mM dNTPs2μL
25mM MgCl20.8μL
10P 5'primer0.6μL
10P 3'primer0.6μL
rTaq0.4μL
dH2O12.6μL
total20μL

temperaturetimecycle
95°C2min
95°C15sec25cycle
55°C30sec25cycle
68°C2min30sec25cycle
68°C2min30sec
14°C∞

10 August

11 August

12 August

13 August

1-11μL
10×M buffer0.5μL
Hind Ⅲ0.2μL
dH2O3.3μL
total5μL

14 August

1-1sample1
10ng/μL TNFAIP36μL
10×KOD plus buffer10μL
2mM dNTPs10μL
25mM MgSO43.2μL
10P 5'primer3μL
10P 3'primer3μL
KOD plus2μL
dH2O62.8μL
total100μL

temperaturetimecycle
95°C2min
95°C15sec25cycle
60°C30sec25cycle
68°C2min30sec25cycle
68°C2min30sec
14°C∞

15 August

16 August

17 August

18 August

19 August

20 August


1. Reproduction of DNA from gel
We did agarose gel electrophoresis (0.7% gel).

Composition
a product of PCR attB TNFAIP3(8/1)
DNA sample90μL
6×Dye18μL
Total108μL

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)

21 August


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 buffer5μL
Total8μL

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.

22 August


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.

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