Team:HokkaidoU Japan/Notebook/RBS SD8

From 2013.igem.org

Maestro E.coli

Notebook

SD8

  • Start

  • Primer Annealing

    Anneal rbs-ensd-f and rbs-ensd-r
    Anneal B0034-f and B0034-r
  • Mini-prep

    plasmid extraction of R0040 on pSB1A2 and LacZα on pSB1AK2
    However, we forgot to add RNAse to mP1. We failed in mini-prep. cry...
  • PCR

    B0015 100up, 200dn, KOD Plus NEO
  • Mini-prep

    plasmid extraction of LacZα on pSB1AK2
  • Digestion

    Cut LacZ%alpha; SpeI and PstI(using 10×H buffer), B0015 fragment by XbaI and PstI(using 10×M buffer)
  • Ligation

    Ligate RBS with R0040
  • Gel Extraction

    LacZα
  • Ethanol Precipitation

    LacZα
  • Transformation

    R0040-RBS (SD8/ B0034) on pSB1A2
    each of 1µL DNA to DH5α
    faild
  • Digestion

    B0015(B0015)
    faild
  • Digestion

    Cut B0015 XbaI and PstI (using 10×M buffer)@
  • Transformation

    R0040-RBS (SD8/ B0034) on pSB1A2
    each of 1µL DNA to JM109
  • Colony PCR

    R0040-(SD8,B0034)
  • Culture

    R0040-(SD8,B0034)
  • Digestion

    Cut B0015(B0015) XbaI and PstI (using 10×M buffer)
  • Ligation

    Ligate LacZαwith B0015
  • Transformation

    LacZα-B0015 on pSB1A2
    each of 1µL DNA to DH5α
  • Colony PCR

    R0040-SD8 in JM109
  • Culture

    R0040-RBS(SD8x2)
  • Mini-Prep

    R0040-RBS(B0034)
  • Sequencing

    R0040-RBS(B0034)
    100bup or 200bdn

    Faild
  • Mini-Prep

    R0040-RBS(SD8)
  • Colony PCR

    LacZα-B0015
  • Culture

    LacZα-B0015
  • Mini-Prep

    LacZα-B0015
  • Sequencing

    R0040-RBS(B0034)
    100bup or 200bdn
    faild
  • Digestion

    Cut R0040-RBS(SD8) SpeI and PstI (using 10×H buffer) LacZα-B0015 XbaI and PstI (using 10×M buffer)
  • Gel Extraction

    R0040-SD8, LacZα-B0015
  • Ethanol Precipitation

    R0040-SD8, LacZα-B0015
  • Liquid culture

    R0040-B0034
  • Sequencing

    R0040-RBS(B0034)
    100bup or 200bdn
    faild
  • Ligation

    Ligate R0040-RBS(SD8) with LacZα-B0015
  • Mini-Prep

    R0040-B0034
  • Transformation

    R0040-RBS(SD8)-LacZα-B0015
    each of 5µL DNA to DH5α
  • Colony PCR

    LacZα-B0015-R0040-RBS(SD8)
  • Liquid culture

    R0040-SD8
  • Digestion

    Cut R0040-SD8 SpeI and PstI (using 10×H buffer) LacZα-B0015 XbaI and PstI (using10×M buffer)
  • Gel Extraction

    R0040-SD8 and LacZα-B0015
  • Ethanol Precipitation

    R0040-SD8 and LacZα-B0015
  • Ligation

    Ligate R0040-RBS(SD8) with LacZα-B0015
  • Colony PCR

    R0040-SD8-LacZα-B0015 on 1A2 in DH5α (yesterday we transformed again)
  • Transformation

    R0040-SD8-LacZα-B0015
  • Mini-prep

    R0040-SD8-LacZα-B0015
  • Sequencing

    R0040-RBS(SD8/B0034)-LacZα-B0015 100up 200dn
  • Sequencing

    R0040-RBS(SD8/B0034)-LacZα-B0015 100up 200dn
  • β-galactosidase assay (1)

    R0040-RBS(B0034/SD8)-LacZα-B0015, R0040-B0034 (as N. C.)
  • β-galactosidase assay (2)

    R0040-RBS(B0034/SD8)-LacZα-B0015, R0040-B0034 (as N. C.)
  • β-galactosidase assay (3)

    R0040-RBS(B0034/SD8)-LacZα-B0015, R0040-B0034 (as N. C.)
  • β-galactosidase assay (4)

    R0040-RBS(B0034/SD8)-LacZα-B0015, R0040-B0034 (as N. C.)
  • β-galactosidase assay (5)

    R0040-RBS(B0034/SD8)-LacZα-B0015, R0040-B0034 (as N. C.)
  • Complete!