Team:KIT-Kyoto/Notebook-week3

August 20th

1. Isolation of DNA fragment from the gel Sample applied to the electrophoresis

Composition

	a product of PCR attB TNFAIP3(8/1)
DNA sample	90μL
6×Dye	18μL
Total	108μL

Results



DNA was isolated from the agarose gel by QIA Quick Gel Extraction Kit, then the DNA was dissolved in 40 uL of TE.

August 21st

1. Measuring the concentration of the attB TNFAIP3 DNA fragment
The order of sample applied to the electrophoresis
1kbmarker3uL，1uL of 5 fold dilution of attB TNFAIP3, 1uL of 10 fold dilution of attB TNFAIP3 DNA fragment，2uL of 1kbmarker, 1uL of 15 fold dilution of attB TNFAIP3 DNA fragment, 1uL of 20 fold dilution of attB TNFAIP3 DNA fragment, 1kbmarker1uL,

Result



The concentration of the isolated attB TNFAIP3 DNA fragments was estimated to be 35ng/uL.



2. BP reaction
　1.5mL tube

attB TNFAIP3(35ng/μL)	2μL
pDONR(150ng/μL)	1μL
TE buffer	5μL
Total	8μL

We added 2uL of BP Clonase Ⅱ enzyme mix to this solution and incubate it for 2 hour.

3. Transformation
　We added 100uL of XL1-Blue to the BP reaction products to do transformation.

August 22nd

We isolated 6 colonies from a plate and incubated in 2.5mL of Kanamycin(＋) LB liquid culture medium for 16 hours.

August 23rd

1 Purification of the candidate pENTR-TNFAIP3 DNA
We purified the candidate pENTR-TNFAIP3 DNA fro six independent colonies cultured on August 22 using QIA prep Spin Miniprep

2 Characterization of the candidate pENTR-TNFAIP3 DNA
We conducted PCR on the following condition to confirm the pENTR-TNFAIP3 DNA using primer designed for attB

Composition

BP TNFAIP3	0.2μL
10× rTaq buffer	0.8μL
2mM dNTPs	2μL
25mM MgCl2	2μL
10P 5'primer	0.6μL
10P 3'primer	0.6μL
rTaq	0.4μL
dH2O	13.4μL
Total	20μL

Reaction

temperature	time	cycle
95°C	2min.
95°C	15sec	25cycle
60°C	30sec	25cycle
68°C	2min30sec	25cycle
68°C	2min30sec
14°C	∞

The PCRproducts were applied to agarose gel electrophoresis
From left to right: pDONR DNA 1uL, ６the candidate pENTR-TNFAIP3 2uL each

Photo of agarose gel


The amplified DNA fragments were detected in the gel.

3. LR reaction
LR reactions were carried out under conditions as described below.

pENTR-TNFAIP3 (prepared on 8/23)	1μL
pTFW or pTGW (Destination vectors)	0.5μL
TE Buffer	6.5μL
Total	8μL

2uL LR clonaseⅡ enzyme mix was added to the reaction and incubated for 2.5 hour.

4. The LR reaction products were transformed into E. coli XL1-Blue100uL according to the protocol and spread on the LB ampicillin(+) plate and cultured for 16 hours at 37℃.

August 24th

Single colonies were isolated from a plate with the candidate pTFW-TNFAIP3 and pTGW-TNFAIP3 and cultured them in LB ampicillin(+) liquid medium for 16 hours at 37℃.

August 25th

1. Purification of the candidate pTFW-TNFAIP3 and pTGW-TNFAIP3 DNA
the candidate pTFW-TNFAIP3 and pTGW-TNFAIP3 were purified by QIA prep Spin Miniprep Kit.

2. Characterization of the candidate pTFW-TNFAIP3 and pTGW-TNFAIP3
We used the primer attB to confirm the candidate pTFW-TNFAIP3 and pTGW-TNFAIP3 for PCR reactions under conditions described below.

Composition

pTFW- or pTGW-TNFAIP3	0.2μL
10× rTaq buffer	0.8μL
2mM dNTPs	2μL
25mM MgCl2	2μL
10P 5'primer	0.6μL
10P 3'primer	0.6μL
rTaq	0.4μL
dH2O	13.4μL
Total	20μL

Reaction

temperature	time	cycle
95°C	2min.
95°C	15sec	25cycle
60°C	30sec	25cycle
68°C	2min30sec	25cycle
68°C	2min30sec
14°C	∞

Photo of agarose gel


Results: Since the amplified DNAs with appropriate sizes were detected on the gel, pTFW-TNFAIP3 and pTGW-TNFAIP3 were appeared to be successfully constructed by the Gateway system ! At this point we renamed the pTFW-TNFAIP3 as pUAS-flag-TNFAIP3.

August 26th

The female-virgin flies (w; Δ2,3) and male flies (yw) were collected for microinjection. The collected flies were kept in the 25℃ incubator separately.