Team:KIT-Kyoto/c6h12o6

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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

22 August

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