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Team:KIT-Kyoto/Notebook-week2p
From 2012.igem.org
August 31st1. PCR amplification of DNA fragments containing UAS and Heat Shock promoter We have carried out the PCR reaction for UAS again. PCR amplification of Heat Shock promoter from pCasPer DNA was also carried out in the following reactions.
Reaction conditions
2. Purification of pAct5C DNA and pGaTB DNA pAct5C DNA and pGaTB DNA was purified from E. coli prepared on 8/29by QIA prep Spin Miniprep Kit. September 1st1. Agarose gel electrophoresis of PCR produsts and the purified pAct5C DNA and pGaTB DNA Agarose gel image is shown below. Left to right: HS promoter 10uL, UAS fragment 10uL, pAct5C DNA-1, pGaTB DNA-1, pGaTB DNA-2, pAct5C DNA 
2. Expected bands for pAct5C DNA and pGaTB DNA were detected on the gel, but band for UAS fragment was not. September 3rd1. PCR amplification of DNA fragments containing GAL4, Act5C promoter and LacZ sequence pGaTBDNA was used as a template to amplify GAL4 sequence. pAct5C-LacZ DNA was used to amplify Act5C promoter-enhancer region and LacZ.
Reaction conditions
2. Agarose gel electrophoresis of the PCRproducts Left to right: 1kb ladder marker 5uL GAL4 fragments 10uL Act5C promoter-enhancer 10uL LacZ sequence 
Results: Expected PCR products were all detected on the gel. These PCR products were purified by High Pure PCR Product Kit (Roche). September 4th1. Digestion of GAL4 fragments by XbaⅠ and SpeⅠ
2. Digestion of pSB1C3 DNA, LacZ fragments and EGFP fragments with BglⅡ pSB1C3 DNA prepared on 8/27, LacZ fragment prepared on 9/3 and EGFP fragments were digested with BglⅡ in the following conditions. 3. Digestion of pSB1C3 DNA with XbaⅠ and SpeⅠ pSB1C3 DNA (vector) prepared on 8/23 was double digested with XbaⅠ and SpeⅠ.
Digestion of pSB1C3 DNA with XbaⅠ and SpeⅠ pSB1C3 DNA (vector) prepared on 8/23 was double digested with XbaⅠ and SpeⅠ.
4. Purification of GAL4 fragments After agarose gel electrophoresis, the digested GAL4 fragments were purified by QIA quick Gel Extraction Kit Photo of agarose gel 
5. Purification of BglII-digested LacZ fragments BglII-digested LacZ fragments were purified by High Pure PCR Product Kit. September 5th1. Purification of EGFP fragments and HS promoter fragments EGFP fragments prepared on 8/29 and HS promoter fragments prepared on 8/31were purified by High Pure PCR Product Kit 2. EGFP fragments and pSB1C3 DNA prepared on 8/27were digested with BglⅡ in the following reaction.
3. Purification of pSB1C3DNA digested with XbaⅠ and SpeⅠ Agarose gel electrophoresis image of the purified sample are shown below. 
4. SpeI digestion of the BglⅡ-digested LacZ, EGFP, pSB1C3 DNA SpeⅠ digestion was carried out in the following reactions.
5. Ligation of pSB1C3 DNA and GAL4 fragment Ligation was carried out in the following reactions.
6. The ligated products were transformed into E. coli XL-1 Blue and spread on the LB Chloramphenicol(＋) plate 7. Purification of pSB1C3, EGFP, LacZfragments double digested with BglⅡ and SpeⅠ The double digested samples were applied to the agarose gel electrophoresis and the DNA fragments were purified by QIA quick Gel Extraction Kit Photo of agarose gel 
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 |
