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Team:KIT-Kyoto/Notebook-week2
From 2012.igem.org
August 8thWe isolated 5 colonies from the LB plate, and cultured. August 9th1. Purification of the candidate pUAS-flag-TNFAIP3 DNA The candidate pUAS-flag-TNFAIP3 DNAs from five independent sample -1,-2,-3,-4 and -5 were purified by the alkaline-lysis method. We added 0.2uL of 10ng/uL RNase A to them and incubate them for 1 hour. 2. Characterization of the candidate pUAS-flag-TNFAIP3 DNA The candidate pUAS-flag-TNFAIP3 DNA was digested wit EcoRⅠ as follows.
After incubation of these samples for 1hour, we added 1uL of 6×Dye to each of them and electrophoresed in 0.7% agarose gel in following order. Left, 10fold dilution, 2uL marker, 20fold dilution, 30fold dilution, 1uL marker, 40fold dilution, 0.5uL marker right. Result 
The concerntration of the candidate pUAS-flag-TNFAIP3 DNA was estimated as 200ng/uL. 3. PCR amplification of the insert DNA The four 5'primers were designed for PCR as follows. ・GW3r 5’-ATCGAGGCCTGTCTAGAGAAGC ・TNFA-1 5’-GGCTTTGCTATGATACTCGGAACTG ・TNFA-2 5’-GTAAAATGTGAAACGCCCAACTGC ・TNFA-3 5’-GGACTCCAGAAAACAAGGGCTTT The 3’ primer was designed as follows. ・SVr 5’-GGCATTCCACCACTGCTCCC PCR reactions with the following combinations of primers were carried out. sample１→GW3r―SVr sample２→TNFA-1―SVr sample３→TNFA-2―SVr sample４→TNFA-3―SVr PCR was carried out in the following reactions.
Reaction conditions of PCR
August 10thElectrophoresis of PCR products was carried out. After adding 2uL of 6×Dye to each 10uL of sample, we electrophoresed them in 0.7% agarose gel in following order. Left marker5uL sample１ sample２ sample３ sample４ Wright Result 
The sizes of the PCR products were unexpected ones. We therefore concluded the construction of pUAS-flag-TNFAIP3 DNA was not successful. August 13thCut check of the plasmid DNA(the candidate pENTR-TNFAIP3) after BP reaction. The candidate pENTR-TNFAIP3 was digested with HindⅢ that will cut out the insert DNA.
After reaction, samples were electrophoresed in following order. Left: 1kb marker5uL cut sample Right: uncut sample Results 
Insert size was different from the expected one. We therefore concluded that the construction of entry DNA was not successful. August 14thBased on the results, we decided to carry out the PCR reaction for TNFAIP3 DNA again. Composition of the reaction
Reaction conditions
PCR product was applied to the 0.7% agarose gel electrophoresis. Results 
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