Team:KIT-Kyoto/c6h12o6

August 1st

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.

(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

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.

August 3rd

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

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

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

E.coli carrying the plasmid sample -1 and -2 in LB Kanamycin(+)liquid culture medium for 16 hours.

August 7th

①Purification of candidate pENTR-TNFAIP3
We purified candidate plasmid-1 and -2 by QIA Prep Spin Miniprep Kit, dissolved it in 30uL of Buffer EB.
Then,the samples were electrophoresed in 0.7% agarose gel.

We made samples as follows.

	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

We electrophoresed them in following order.
Left marker 5uL control pDONR DNA ,pENTR-TNFAIP3-1 DNA,pENTR-TNFAIP3 -2 DNA marker 4uL marker 3uL Wright.

Results

Concentration of pENTR-TNFAIP3-2 DNA was estimated at around 100ng/uL.


②　LR reaction
We made following solution(vials)in 1.5mL tube.

BP TNFA-2	2μL
pTFW(Destination vector)	0.5μL
TE buffer	6.5μL
total	9μL

we added 1uL of LR clonaseⅡ enzyme mix to this solution and incubate it for 2.5 hours.

③ Transformation of E.coli with LR reaction products.
Transformation of E.coli was carried out by adding 2uL of LR reaction products to 100uL of XL1-Blue and plated on the LB ampicillin(+)plate and cultured at 37°C.


WEEK１終わり

August 8th

We isolated 5 colonies from the LB plate, and cultured.

August 9th

①Purification of the candidate pUAS-flag-TNFAIP3 DNA

The candidate pUAS-flag-TNFAIP3 DNAs from five independent sample -1,-2,-3,-4 and -5 were purified by the alkaline-lysis method.
We added 0.2uL of 10ng/uL RNase A to them and incubate them for 1 hour.


②Characterization of the candidate pUAS-flag-TNFAIP3 DNA

The candidate pUAS-flag-TNFAIP3 DNA was digested wit EcoRⅠ as follows.

Each diluted DNA sample	1μL
10×H buffer	0.5μL
EcoRⅠ	0.2μL
dH2O	3.3μL
total	5μL

After incubation of these samples for 1hour, we added 1uL of 6×Dye to each of them and electrophoresed in 0.7% agarose gel in following order.
Left,　10fold dilution,　2uL marker,　20fold dilution,　30fold dilution,　1uL marker,　40fold dilution,　0.5uL marker right.


Results


The concerntration of the candidate pUAS-flag-TNFAIP3 DNA was estimated as 200ng/uL.


③ PCR amplification of the insert DNA

The four 5'primers were designed for PCR as follows.

・GW3r
5’-ATCGAGGCCTGTCTAGAGAAGC
・TNFA-1
5’-GGCTTTGCTATGATACTCGGAACTG
・TNFA-2
5’-GTAAAATGTGAAACGCCCAACTGC
・TNFA-3
5’-GGACTCCAGAAAACAAGGGCTTT


The 3’ primer was designed as follows.
・SVr
5’-GGCATTCCACCACTGCTCCC


PCR reactions with the following combinations of primers were carried out.
sample１→GW3r―SVr
sample２→TNFA-1―SVr
sample３→TNFA-2―SVr
sample４→TNFA-3―SVr


PCR was carried out in the following reactions.

	Each sample
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

Reaction conditions of PCR

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

Electrophoresis of PCR products was carried out.
After adding 2uL of 6×Dye to each 10uL of sample, we electrophoresed them in 0.7% agarose gel in following order.

Left　marker5uL　sample１　sample２　sample３　sample４　Wright

Results

The sizes of the PCR products were unexpected ones. We therefore concluded the construction of pUAS-flag-TNFAIP3 DNA was not successful.

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.