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Team:KIT-Kyoto/Notebook-week1p
From 2012.igem.org
August 23rdTransformation of E. coli with pEGFP-C2 DNA and pAct5C-GAL4 DNA E. coli transformed with pEGFP-C2 was spread on the LB Kanamycin(＋) plate and that transformed with pAct5C GAL4 was spread on the LB ampicillin(＋) plate. August 24thThe appropriate colonies were picked up and cultured in the LB medium containing the appropriate antibiotics. August 25th1. Purification of the pEGFP-C2 DNA and the pAct5C-GAL4 DNA These plasmid DNAs were purified from E. coli by QIA prep Spin Miniprep Kit. 2. Digestion of pEGFP-C2 DNA with BamHⅠ and BglⅡt Restriction enzyme digestion was carried out in the following reaction
3. Agarose gel electrophoresis of the digested DNA The digested DNA was applied to the agarose gel electrophoresis and linearized DNA was purified by QIA quick Gel Extraction Kit. Photograph of the agarose gel 
4. Digestion of pSB1C3DNA with EcoRⅠ and SpeⅠ pSB1C3 DNA was digested with EcoRⅠ and SpeⅠ at 37℃ for 16 hours.
5. PCR amplification of the UAS region and pSB1C3 DNA DNA fragments containing UAS was amplified from the pUAST DNA and the BglⅡ site-containing pSB1C3 was also amplified by PCR.
Reaction conditions
August 26th・Purification of the digested pSB1C3 pSB1C3 DNA double-digested with EcoRⅠ and SpeⅠ(prepared on 8/25) was applied to the agarose gel electrophoresis. The DNA fragment of interest was purified from the gel by QIA quick Gel Extraction Kit. Photo of the agarose gel 
August 27th1. Removing the multi-cloning site of the pEGFP-C2 pEGFP-C2 DNA digested with BglⅡ and BamHⅠwas self ligated in the following reaction.
2. The ligated products were transformed into the E. coli XL-1 blue and the E. coli was apreaded on the LB Kanamycin(＋) plate and cultured at 37℃ for 16 hours. 3. Agarose gel electrophoresis of the DNA fragments containing UAS and the PSR products from the pSB1C3 PCRproducts prepared on August 25th were applied to the agarose gel electrophoresis as shown below. Photo of the agarose gel 
Since the expected DNA fragments were detected, DNA was purified from the agarose gel by QIA quick Gel Extraction Kit. The purified DNAs were applied to the agarose gel electrophoresis as shown below. Photo of the agarose gel 
August 28th1. Single colony isolation from the E. coli transformed with pEGFP-C2 DNA Single colony isolationwas carried out from the plate prepared on 8/27and cultured in 2.5mL LB Kanamycin(＋) medium at 37℃ for 16 hours. 2. Transformation of E. coli with pGaTB DNA or pAct5C-GAL4 DNA E. coli Xl-1 blue was transformed with pGaTBDNA or pAct5C-GAL4 DNA and cultured on LB ampicillin(＋) plate for 16 hours at 37℃. August 29th1. Single colony isolation from the E. coli transformed with pGaTB DNA or pAct5C-GAL4 DNA From the plate prepared on 8/28 single colonies were isolated and cultured in 2.5mL LB ampicillin(＋) medium for 16 hours at 37℃. 2. Purification of pEGFP-C2 DNA pEGFP-C2 DNA was purified from E. coli prepared on 8/28 by QIA prep Spin Miniprep Kit. 3. Cut check of the pEGFP-C2 DNA The purified pEGFP-C2 was digested with XhoⅠ and PstⅠ in the following reactions.
The digested samples were applied to the agarose gel electrophoresis.as shown below. Photo of the agarose gel 
Results: The DNAs were undigested by these enzymes, confirming that the multi-cloning site of the pEGFP-C2 was successfully removed. 4. PCR amplification of the DNA fragments containing UAS and EGFP Since we found that the previously used primers for PCR were wrong, PCR amplification of the DNA fragments containing UAS and EGFP region were carried out in the following reactions.
Reaction conditions
Agarose gel electrophoresis of the PCR products 
Results: DNA fragments containing EGFP were successfully amplified, but not for the UAS. |
