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August 20th

1. Reproduction of DNA from gel
We did agarose gel electrophoresis (0.7% gel).

a product of PCR attB TNFAIP3(8/1)
DNA sample90μL

We separated 54uL×2 from 108uL (sample) and we did electrophoresis (54uL sample and 1kb marker 5uL).We separated gel containing DNA.

We isolated DNA from these gels by QIA quick Gel Extraction Kit.
Finally we melt DNA to TE Buffer(40uL)

August 21st

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 )

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

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.

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.