Team:KIT-Kyoto/c6h12o6

August 1st

	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

	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

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

August 5th

August 6th

August 7th

	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

August 9th

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

August 11th

August 12th

August 13th

1-1	1μL
10×M buffer	0.5μL
Hind Ⅲ	0.2μL
dH2O	3.3μL
total	5μL

August 14th

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

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

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

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.