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