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Team:Cambridge/Protocols/MillerAssay
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*Take suspension of cells grown up overnight and dilute to a cell density of 0.1-0.2 in appropriate growth medium (for example LB broth for e.coli). If genetic construct can be selected for by an antibiotic, include this in the growth medium according to needs - this prevents loss of the plasmid during growth. | *Take suspension of cells grown up overnight and dilute to a cell density of 0.1-0.2 in appropriate growth medium (for example LB broth for e.coli). If genetic construct can be selected for by an antibiotic, include this in the growth medium according to needs - this prevents loss of the plasmid during growth. | ||
| - | *Now add the following to each of your | + | *Now add the following to each of your eppendorf tubes: |
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||Z buffer (60mM Na<sub>2</sub>HPO<sub>4</sub>.7H<sub>2</sub>O, 40mM NaH<sub>2</sub>PO<sub>4</sub>.H<sub>2</sub>O, 10mM KCl, 1mM MgSO<sub>4</sub>.7H<sub>2</sub>O, 50mM β-mercaptoethanol)||1ml | ||Z buffer (60mM Na<sub>2</sub>HPO<sub>4</sub>.7H<sub>2</sub>O, 40mM NaH<sub>2</sub>PO<sub>4</sub>.H<sub>2</sub>O, 10mM KCl, 1mM MgSO<sub>4</sub>.7H<sub>2</sub>O, 50mM β-mercaptoethanol)||1ml | ||
|- | |- | ||
| - | ||Freshly prepared 0.1% SDS|| | + | ||Freshly prepared 0.1% SDS||20μl |
|- | |- | ||
||Chloroform||40μl | ||Chloroform||40μl | ||
Revision as of 14:39, 20 September 2012
Miller Assay
Protocol
- Take suspension of cells grown up overnight and dilute to a cell density of 0.1-0.2 in appropriate growth medium (for example LB broth for e.coli). If genetic construct can be selected for by an antibiotic, include this in the growth medium according to needs - this prevents loss of the plasmid during growth.
- Now add the following to each of your eppendorf tubes:
| Reagent | Volume |
| Z buffer (60mM Na2HPO4.7H2O, 40mM NaH2PO4.H2O, 10mM KCl, 1mM MgSO4.7H2O, 50mM β-mercaptoethanol) | 1ml |
| Freshly prepared 0.1% SDS | 20μl |
| Chloroform | 40μl |
| Cell suspension | 100μl |
- Vortex all the tubes for 10s
- Allow the chloroform to settle to the bottom of the tubes and then transfer 100μl into each well along a row in a 96 well plate. You can reliably get 9 aliquots out of the reaction volume given above, i.e. you can test 9 different conditions. Be sure to leave some medium in the tube, if you transfer through any of the chloroform it will melt through your microarray plate. If more than 9 aliquots are required then add more than 1ml of Z buffer.
- Add 20μl of 4mg/ml ONPG to each well. This should be made fresh in 0.1M sodium phosphate buffer
- Add the desired concentrations of test substance to each well and place in the plate reader.
- Ideally you wish to record the A600 (to correct for cell density), A420 (the value you are interested in), and A550 (a correction factor for light scattering). This allows you to calculate the Miller units for your assay using this formula: Miller Units = 1000 x [(OD420 - 1.75 x OD550)] / (Time of reaction x Reaction volume in ml x OD600). However, if you can only measure one parameter then A420 on its own will give a good indication of the assay's outcome.
- Take readings every 15 minutes over ~10 hours, the exact time is dependent on the rate of induction of β-galactosidase by your test substance.
