Difference between revisions of "Template:BioE140LSpr09-Growth"
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* Heat shock for 2 min at 42 | * Heat shock for 2 min at 42 | ||
* Put back on ice for 1 min | * Put back on ice for 1 min | ||
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* Add 100uL of LB, let shake in the 37 degree incubator for 40 min | * Add 100uL of LB, let shake in the 37 degree incubator for 40 min | ||
* Plate on selective antibiotics, let incubate overnight | * Plate on selective antibiotics, let incubate overnight | ||
Revision as of 00:14, 11 May 2009
Growth Curve Assay
The purpose of this assay is to determine the toxicity of various composite constructs. These constructs include:
M10210 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPG_L6!}{dblTerm}
M10211 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<eCPX!}{dblTerm}
M10212 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<upaG_short!}{dblTerm}
M10213 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<Ag43_short!}{dblTerm}
M10214 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<espP(beta)!}{dblTerm}
M10215 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<ehaB!]{dblTerm}
M10216 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPompX!}{dblTerm}
M10217 {Pbad.rbs.prepro.StrepTag}{<AG4>}{<TshA!}{dblTerm}
M10218 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<CPG_L6!}{dblTerm}
M10219 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<eCPX!}{dblTerm}
M10220 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<upaG_short!}{dblTerm}
M10221 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<Ag43_short!}{dblTerm}
M10222 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<espP(beta)!}{dblTerm}
M10223 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<ehaB!]{dblTerm}
M10224 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<CPompX!}{dblTerm}
M10225 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<TshA!}{dblTerm}
With all the constructs under an arabinose promoter, toxicity can be inferred from the differences in growth rate (as determined by changes in OD 600 over time) between samples containing LB and LB+arabinose.
Procedures
Plating
The controls are: DH10B and pBca9495CA-Bca1144 (as controls, the DH10B is blank control and pBca9495CA-Bca1144 controls for plasmid effects.)
- Take 2 tubes of 280ul of cell and add 60ul KCM and 100ul water to each.
- Add 20ul of the KCM/water/cell solution into each construct.
- Transform (see Transformation section below)
- Grow plates overnight.
- Pick 5 colonies from each sample.
- Grow to saturation in 96 well blocks with 400 uL LB media in each well.
- Then from each of the 5 unique liquid cultures, make an arabinose sample and a non-arabinose sample.
- Add 50 uL of LB media or LB Media+100ug/mL arabinose per well in 384 well plate.
- Add 1 uL of cell sample to each well.
- Place plate in Tecan and run OD measurements every 10 minutes.
Transformation
- Thaw 2 tubes of 280 uL aliquot of cells on ice
- Add 100 uL of water to each tube
- Add 60 uL of KCM salts to each tube
- Add 1ul of the constructs to 20 uL of the cell cocktail. Pipette up and down gently to mix
- Let sit on ice for 10 min
- Heat shock for 2 min at 42
- Put back on ice for 1 min
- Add 100uL of LB, let shake in the 37 degree incubator for 40 min
- Plate on selective antibiotics, let incubate overnight