Difference between revisions of "Team:Dundee/pmodal"
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<ol> | <ol> | ||
− | <li> | + | <li> Take 3 independent colonies from each plate of your controls and samples and place each colony into 5 ml of LB with the appropriate antibiotics. </li> |
− | <li> | + | <li> Leave to grow overnight at 30°C.</li> |
− | <li> | + | <li> Take 50 μl of each overnight and inoculate 5 ml of fresh LB containing the appropriate antibiotics. Do this twice for every independent colony. </li> |
− | <li> | + | <li> Allow these to grow at 30°C shaker until the O.D of each inoculate is between 0.3-0.5</li> |
− | <li> | + | <li> Once the O.D is between 0.3-0.5 take 3 x 1 ml samples of each culture and place into separate Eppendorf tubes.</li> |
− | <li> | + | <li> Take a further 1 ml and place into a cuvette and subsequently measure the OD<sub>600</sub> marking down the OD for each replica. </li> |
− | <li> | + | <li> Spin down the 1 ml cultures of each replica at 13,000 rpm for 3 minutes. </li> |
− | <li> | + | <li> Remove the supernatant from each 1 ml culture and freeze cell pellet for later use in the β-galactosidase Assay. </li> |
</ol> | </ol> | ||
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<p> | <p> | ||
<ol> | <ol> | ||
− | <li> | + | <li> Resuspend cell pellet in 800 μl of buffer 2 (100 mM dibasic sodium phosphate (Na2HPO4); 20 mM KCl ; 2 mM MgSO4 ;0.8 mg/mL CTAB (hexadecyltrimethylammonium bromide) ; 0.4 mg/mL sodiumdeoxycholate) and subsequntly add 5.4 μL/mL beta-mercaptoethanol. </li> |
− | <li> | + | <li> Add 50 μl of SDS (0.1%) to each sample. </li> |
− | <li> | + | <li> Add 50 μl of Chloroform to each sample and vortex for 30 seconds. </li> |
− | <li> | + | <li> Make substrate solution (60 mM Na2HPO4; 40 mM NaH2PO4; 1 mg/mL o-nitrophenyl-β-D-Galactoside (ONPG); 2.7 μL/mL β-mercaptoethanol)</li> |
− | <li> | + | <li> Add 200 μl of substrate solution to each sample marking down the time it was added in relation to the first sample. </li> |
− | <li> | + | <li> Wait for an observable color change and mark down the time that the color change occurred.</li> |
− | <li> | + | <li> Add 600 μl of NaCO3(1M) once the color change has occurred.</li> |
− | <li> | + | <li> Spin down the samples at 13,000 rpm for 3 minutes and place the supernatant into a cuvette. </li> |
− | <li> | + | <li> Measure the OD<sub>420</sub>. </li> |
</ol> | </ol> | ||
</p> | </p> |
Revision as of 15:03, 17 September 2015
- Resuspend cell pellet in 800 μl of buffer 2 (100 mM dibasic sodium phosphate (Na2HPO4); 20 mM KCl ; 2 mM MgSO4 ;0.8 mg/mL CTAB (hexadecyltrimethylammonium bromide) ; 0.4 mg/mL sodiumdeoxycholate) and subsequntly add 5.4 μL/mL beta-mercaptoethanol.
- Add 50 μl of SDS (0.1%) to each sample.
- Add 50 μl of Chloroform to each sample and vortex for 30 seconds.
- Make substrate solution (60 mM Na2HPO4; 40 mM NaH2PO4; 1 mg/mL o-nitrophenyl-β-D-Galactoside (ONPG); 2.7 μL/mL β-mercaptoethanol)
- Add 200 μl of substrate solution to each sample marking down the time it was added in relation to the first sample.
- Wait for an observable color change and mark down the time that the color change occurred.
- Add 600 μl of NaCO3(1M) once the color change has occurred.
- Spin down the samples at 13,000 rpm for 3 minutes and place the supernatant into a cuvette.
- Measure the OD420.