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 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.