User:David.lim.yale/17 July 2012

From 2012.igem.org

Contents

Make glycerol stocks of Pedraza-Reyes strains

  • Aliquot 600 mL of the O/N cultures of PERM311, PERM704, and PERM739 into Eppendorf tubes
  • Add 200 mL 60% glycerol solution to each tube
  • Store at -80o C

Make stock solution of 2mg/mL L-Trp

  • Measure out 100 (99.91) mg of L-Trp
  • Bring to 50 mL with dH2O
  • Dissolve solids by mixing gently while heating
  • Filter sterilize
  • Store at RT with foil covering tube (Trp is light sensitive)

Make stock solution of 1% (w/v) casein hydrolysate

  • Measure out 500 (501.2) mg of casein hydrolysate
  • Bring to 50 mL with dH2O
  • Dissolve solids by mixing gently
  • Filter sterilize
  • Store at RT

Make stock solution of 0.1 M EGTA (pH 8.0)

  • Measure out 5.70 (5.71) g of EGTA
  • Add NaOH pellets and 100 mL dH2O to dissolve EGTA
  • Add 2N HCl until pH is at 8.0 (approximated using 5.0-10.0 pH paper)
  • Bring to 150 mL
  • Filter sterilize
  • Store at RT

Make O/N cultures (3x) of pBR322-transformed Ec XL1-Blue competent cells

  • Add 2 μL of 100mg/mL Amp stock solution to 2 mL LB culture tubes
  • Inoculate a single colony from XL1-blue transformants per tube
  • Incubate overnight at 34o C
left: XL1-blue cells transformed with 2 μL of sterile dH2O
right: XL1-blue cells transformed with 2 μL of 1:10 pBR322 from Isaacs lab