Revision as of 02:47, 25 September 2012 by Kazuko (Talk | contribs)

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 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.


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 23rd

August 24th

August 25th

 All DNA  500ng  1ug 
 pEGFP-C  3uL  6uL 
 H buffer(TOYOBO)  5uL  5uL 
 BamHⅠ(TOYOBO)  1uL  1uL 
 BglⅡ(TOYOBO)  1uL  1uL 
 100×BSA  0.5uL  0.5uL 
 dH2O  39.5uL  36.5uL 
 Total  50uL  50uL 

 DNA sample  23uL 
 4 buffer(NEB)  5uL 
 EcoRⅠ-HF(NEB)  1uL 
 SpeⅠ(NEB)  1.5uL 
 100×BSA  0.5uL 
 dH2O  19uL 
 Total  50uL 

 All samples 
 DNA template  0.5uL 
 10× KOD plus buffer  10uL 
 2mM dNTPs  10uL 
 25mM MgSO4  3.2uL 
 10P 5’ primer  3uL 
 10P 3’ primer  3uL 
 KOD plus  2uL 
 dH2O  68.3uL 
 Total  100uL 

 Temperature  Time  Circle 
  95℃ 2min
  95℃ 15sec 25 cycle 
  58℃ 30min(UAS) or 2min10sec(pSB1C3) 25 cycle 
  68℃ 2min30sec 25 cycle 
  68℃ 2min30sec
  14℃ ∞

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.