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Team:Kyoto/LabWork5
From 2011.igem.org
Lab Work Week5: Monday 29th August - Sunday 4th September
Monday
Tuesday
Nutritional Signal(Hashiya)
・PCR amplification of glnL and glnG from PCR products,glnL1 and glnG1.
--Primers--
glnL
Left primer: ggaattcgcggccgcttctagaggagactgctttatggcaac
Right primer: ggactagtaggaactatcgtcatcgactac
glnG
Left primer: ggaattcgcggccgcttctagaggtgacgtttatgcaacga
Right primer: ggactagtacacacaagctgtgaatcactc
annealing temperature was 55 degrees.
・PCR amplification of glnL+G and rpoN from gDNA of E.coli.
--Primers--
glnL+G
Left primer: ggaattcgcggccgcttctagaggagactgctttatggcaac
Right primer: ggactagtacacacaagctgtgaatcactc
rpoN
Left primer: ggaattcgcggccgcttctagaggttctgaacatgaagcaa
Right primer: ggactagtatccttatcggttgggtca
annealing temperature was 56 degrees.
lane1: 100bp DNA ladder, lane2:glnL, lane3:glnG, lane4:glnG+L, lane5:rpoN from gDNA, lane6:rpoN from ASKA clone
After purification, the concentration of DNA are
glnL: 122.3 ng/ul
glnG: 64.7 ng/ul
glnL+G: 106.7 ng/ul
rpoN from gDNA: 111.4 ng/ul
Wednesday
Nutritional Signal(Hashiya)
・Screening PCR of σ54 promoter + pSB1A3
We cultured σ54 promoter5
Friday
Nutritional Signal(Hashiya)
・Restriction of σ54 promoter5,glnL, glnG, glnL+G and rpoN
Cut them with EcoRl and Spel
After purification, the concentration of DNA were
σ54 promoter5: 23.6 ng/ul
glnL: 28.1 ng/ul
glnG: 26.3 ng/ul
glnL+G: 15.3 ng/ul
rpoN: 20.8 ng/ul
・Ligation reaction
Ligated glnL, glnG and rpoN to pSB1K3.
Thursday
Nutritional Signal(Hashiya)
・Screening PCR of glnL, glnG, glnL+G and rpoN
glnL
glnG
glnL+G & rpoN
We cultured glnL5, glnG4
Saturday
Nutritional Signal(Hashiya)
・Mini prep of glnL5 and glnG4
glnL5: 43.9 ng/ul
glnG4: 38.1 ng/ul
・Screening PCR of rpoN
Predation(Hashiya)
・PCR amplification of glmS
--Primers--
left primer:ggaattcgcggccgcttctagagcaggttgaccgacaacgata
right primer:ggactagtacgcagggcatccatttat
lane1: 100bp DNA ladder, lane2: glmS from gDNA, lane3: glmS from ASKA clone
・TA cloning of glmS
Ligated glmS from gDNA to pTA vector.
Sunday
Nutritional Signal(Hashiya)
・Screening PCR of rpoN
・Transformation of bellow parts
1-23L: BBa_B0015 (double terminator)
1-18E: BBa_J23101 (constitutive promoter)
1-18C: BBa_J23100 (constitutive promoter)
