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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 ℃. 1. Single colony isolation of ligationproducts Ligation product prepared on 9/9 (UAS and pSB1C3) gave us some colonies and negative control sample gave us no colony. We therefore picked up four independent colonies and cultured them in 2.5mL LB Chloramphenicol (＋) medium at 37℃ for 16 hours. 2. Digestion of HS promoter fragment and Act5C promoter with XbaⅠ and BamHⅠ DNA fragments carrying HS promoter (prepared on 9/5) and those carrying Act5C promoter (prepared on 9/3) were digested with XbaⅠ and BamHⅠ.
Digested fragments were purified from the gel by QIA quick Gel Extraction Kit. Photo of agarose gel 
3. PCR amplification of GAL4 fragments We tried PCR under four different conditions described below.
Reaction conditions
4. PCR products were applied to agarose gel electrophoresis From left to right: 10uL each G-1, G-2, G-3, G-4 Photo of agarose gel 
We found that GAL4 sequence was properly amplified under G-1 and G-2conditions. 6. Digestion of pSB1C3 DNA with EcoRⅠ and SpeⅠ PCR-amplified pSB1C3 DNA was digested with EcoRⅠ and SpeⅠ for 2 hours at 37℃.
7. Digestion of EcoRI-digested UAS fragment by BglⅡ BglⅡ-digestion was carried out for 2 hours at 37℃ under conditions described below.
8. Purification of restriction enzyme-digested pSB1C3 DNA and UAS fragment restriction enzyme-digested pSB1C3 DNA and UAS fragment (prepared on 9/10) were purified by QIA quick Gel Extraction Kit. Photo of agarose gel 
9. PCR-amplified GAL4 fragments were digested with XbaⅠ and SpeⅠ under conditions described below.
The digested DNA was applied to agarose gel electrophoresis as shown below. Photo of agarose gel 
Results: XbaⅠ-digestion of GAL4 fragment gave two DNA fragments, indicating that GAL4 sequence contains XbaⅠ site. 10. Digestion of GAL4 sequence with BglⅡ and SpeⅠ Digestion was carried out st 37℃ for 1hour under the conditions described below.
The digested DNA was purified by High Pure PCR Product Kit was further digested with Spe I at 37℃ for 1hour.
The digested fragments were purified by QIA quick Gel Extraction Kit. Photo of agarose gel 
11. Insertion of GAL4 fragments and HS promoter or Act5C promoter-enhancer and UASfragment and EGFP sequence or LacZ sequence into the pSB1C3DNA Ligation reactions were carried out at 16℃ for 1hour under conditions described below.
12. Ligation products were transformed into E. coli Xl-1 blue, spread on the LB Chloramphenicol(＋) plate and cultured at 37℃for 16 hours. September 11th1. Single colony isolation Single colonies were isolated from the plate prepared on 9/11 and cultured in 2.5mL LB Chloramphenicol(＋) medium at 37℃ for 16 hours. 2. Isolation of the candidate pSB1C3-UAS DNA The candidate pSB1C3-UAS DNA was purified by QIA prep Spin Miniprep Kit. 3. The purified candudate pSB1C3-UAS was digested with BglII for 2 hours.
The digested samples were applied to agarose gel electrophoresis as shown below. Left to right: pSB1C3-1 uncut, pSB1C3 cut, -2 uncut, -2 cut, -3 uncut, -3 cut, -4 uncut, -4 cut Photo of agarose gel 
Results: All DNAs examined had a single BglII site, indicating that the pSB1C3-UAS DNA was successfully constructed. The BglII-digested pSB1C3-UAS DNA was purified from the gel by QIA quick Gel Extraction Kit. 4. Digestion of pSB1C3 (PCR) with XbaⅠ and SpeⅠ PCR-amplified pSB1C3 DNA was double digested with XbaⅠ and SpeⅠ at 37℃ for 2 hours.
The digested samples were purified by QIA quick Gel Extraction Kit Photo of agarose gel 
5. Insertion of GAL4, HS promoter or Act5C promoter-enhancer into the pSB1C3DNA Ligation reactions were carried out under conditions described below at 16℃ for 1hour .
13. Ligation products were transformed into E. coli Xl-1 blue, spread on the LB Chloramphenicol(＋) plate and cultured at 37℃for 16 hours. 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℃. |
