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Team:KIT-Kyoto/Notebook-week3p
From 2012.igem.org
September 6thTransformation performed on September 5th was not successful, since we had no colony on the plate. 1. PCR amplification of UAS , UAS-TNFAIP3, pSB1C3DNA
Reaction conditions
2. PCR products were applied to the agarose gel electrophoresis From left to right: UAS, UAS-TNFAIP3, pSB1C3 3. Purifucation of pSB1C3 PCR products pSB1C3 PCR products were purified from the gel by QIA quick Gel Extraction Kit. 
4. Ligation of pSB1C3 DNA and GAL4 fragment
5. The ligation products were transformed into E. coli XL-1and spread on the LB Chloramphenicol(＋) plate September 7th1. PCR amplification of UAS, UAS-TNFAIP3, pSB1C3, GAL4 DNA
Reaction conditions
2. PCR products were applied to the agarose gel electrophoresis From left to right: UAS, UAS-TNFAIP3, pSB1C3, GAL4 as shown below Results: no clearly amplified bands were detected. 3. Re-estimation of the concentration of pSB1C3 and GAL4 DNA used for ligation on 9/5, 6 4. SB1C3 and GAL4 DNA were applied to the agarose gel electrophoresis From left to right: 1kbmarker 5uL, DNA 1uL, 1kb marker 3uL, DNA 0.2uL, 1kbmarker - 2uL, DNA 0.1uL, 1kbmarker 1uL 
From these results, concentration of the pSB1C3 DNA was estimated to be 10ng/uL. GAL4 From left to right: 1kbmarker 5uL, DNA 1uL, 1kb marker 3uL, DNA 0.2uL, 1kbmarker 2uL, DNA 0.1uL, 1kbmarker 1uL 
However no GAL DNA was detected. We lost the sample somewhere by some mistakes. September 8th1. PCR amplification of UAS, UAS-TNFAIP3, pSB1C3 and GAL4 DNA
Reaction conditions
5. PCRproducts applied to the agarose gel electrophoresis From left to right: UAS, UAS-TNFAIP3, pSB1C3, GAL4 10 uL each 
Only the PCR products for pSB1C3 was detectable. We repeated PCR for UAS and GAL4DNA by changing the annealing temperature （-1 indicates 55℃、-2 indicates 58℃）
Reaction conditions
September 9th1. PCRproducts were electrophoreses. Left to right: UAS-1, UAS-2, GAL4-1, GAL4-2 10 uL each 
We finally succeeded the amplification of the UAS fragments. 2. Purification of pSB1C3 and UAS-1-PCR products PCR products were purified by High Pure PCR Product Kit 3. The purified UAS fragments were digested with EcoRⅠ and SpeⅠ in the following reaction
4. The digested UAS DNA was applied to the agarose gel electrophoresis and purified from the gel by QIA quick Gel Extraction Kit 
5. PCR amplification of GAL4
Reaction conditions
6. PCR products were applied to the agarose gel electrophoresis as shown below. 
7. pSB1C3 and UAS fragments were ligated in the following reactions.
The following reaction were carried out as a negative control.
8. 7 ligation products were transformed into E. coli XL-1 Blue and spread on the LB Chloramphenicol(＋) plate and incubated at 37℃ for 16 hours. September 10thAt 20 min after removing the medium, 25 uL of serum free medium containing the 1 uL of siLentfect and 200 ng each of pAct5C-GAL4 DNA and pUAS-EGFP-TNFAIP3 DNA was added to the well. At 4 hours after transfection, the transfection medium was removed and the Schneider’s Drosophila medium containing 10% fetal bovine serum was added to each well. The cells were incubated for 48 hours at 25 ℃.
September 11th
September 12th1-2-7 Purification of the candidate pSB1C3-UAS-EGFP and pSB1C3-UAS-LacZ DNAs We purified the candidate pSB1C3-UAS-EGFP and pSB1C3-UAS-LacZ DNAs by QIA prep Spin Miniprep Kit from E. coli cultured in LB medium for 16 hours (9/11). 1-1-47 Digestion of pSB1C3-UAS DNA by Spe1 We incubated BglⅡ-digested pSB1C3-UAS (9/11) DNA with SpeⅠfor 2 hours. The reaction conditions are shown below. Composition
The digested DNA were applied to agarose gel electrophoresis. We isolated DNA from the gel by using QIA quick Gel Extraction Kit. Photo of Agarose gel. 
1-1-48 and 2-2-6 Ligation DNA fragment carrying EGFP or LacZ was ligated into the BglII and SpeI digested pSB1C3-UAS DNA .for 1hour at 16℃. Ligation reactions are listed below.
We ligated DNA fragment carrying GAL4 and DNA fragments carrying HS promoter or Act5c promoter-enhancer to pSB1C3 DNA for 1hour at 16℃. Composition
1-1-49 and 2-2-7 Transformation of E. coli by Ligation products We did transformation of E. coli Xl-1 blue with each of ligation products. Finally, we spread them on the LB Chloramphenicol(+) plate and incubated for 16 hours at 37℃. |
