Team:KIT-Kyoto/c6h12o6

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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





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

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

WEEK1終わり

August 8th


*TNFA and API2

August 9th


*TNFA and API2

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

August 10th


*TNFA and API2





August 13th


*TNFA and API2

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




August 14th


*TNFA and API2

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




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 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)

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 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.

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.