Team:KIT-Kyoto/Notebook-week1

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.

We performed PCR in the following conditions.

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 to right: 1kb marker(5uL), attB TNFAIP3, attB API2-MALT1-1, -2, -3, -4

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

1. BP 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

1. Purification 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.