Team:UGA-Georgia/Protocols
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<li>Place the microcentrifuge tubes into a shaker at 37C for ~20hrs.</li> | <li>Place the microcentrifuge tubes into a shaker at 37C for ~20hrs.</li> | ||
<li>Remove from the shaker and take to a plate reader to run fluorescence tests.</li> | <li>Remove from the shaker and take to a plate reader to run fluorescence tests.</li> | ||
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</ol> | </ol> | ||
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<li>K2HPO4, 14g/L - 1mL</li> | <li>K2HPO4, 14g/L - 1mL</li> | ||
<li>NaCl, 293g/L - 7.5mL</li> | <li>NaCl, 293g/L - 7.5mL</li> | ||
+ | |||
+ | <tr><td colspan="3"> <h3>Separatory Funnel Extraction of Extracellular Content</h3></td></tr> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | <tr> | ||
+ | <td width="80%" valign="top"> | ||
+ | <p><ol> | ||
+ | |||
+ | <li>Take the ODs of all the cultures to be extracted.</li> | ||
+ | <ul> | ||
+ | <li> These cultures should ideally be in the 0.5-0.9 range. </li> | ||
+ | </ul> | ||
+ | <li>Open the cultures using a de-clamp under the fume hood, relieving the culture of any pressure.</li> | ||
+ | <li>Transfer the culture to a centrifuge ready container</li> | ||
+ | <li>Centrifuge the cultures at 11,100G for 20 minutes, or until cell pellet is formed. </li> | ||
+ | <li>After removing the samples from the centrifuge, carefully pour the supernatant into a separatory funnel, being sure to preserve all of the cell pellet. Place the cell pellet and its container into a -20C freezer for 1 hour, if planning on completing the extraction for cellular content. </li> | ||
+ | <li>Pour 5ml of 9:1 hexane/acetone solution into the separatory funnel, there should be a distinct difference between the water phase layer and the organic phase layer. </li> | ||
+ | <li>Carefully, yet thoroughly, mix the water phase and organic phase by inverting the separatory funnel, letting any built pressure escape by briefly removing the top to the separatory funnel after each inversion. Avoid letting any bubbles form by aggressive inversion. </li> | ||
+ | <li>Allow the water phase and organic phase to settle until a distinct difference is visible again. </li> | ||
+ | <li>Drain the supernatant into a container, leaving only the organic phase in the separatory funnel. This supernatant may be discarded. </li> | ||
+ | <li>Drain all of the organic phase into a beaker and add 1.5g of sodium sulfate. Swirl this mixture around thoroughly until most of the powder has formed into small clumps. </li> | ||
+ | <li>Gently pour the filtered organic phase into a new container without including any of the sodium sulfate. Use a micropipette to transfer any remaining organic phase to the new container, if necessary. </li> | ||
+ | <li>Allow the filtered organic phase to dry under a stream of N2 gas until it reaches <1.5ml, then transfer to a 1.5ml microcentrifuge tube. Allow to continue drying under a stream of N2 gas until it reaches 0.1ml, then transfer to a GC/MS sample bottle. </li> | ||
+ | |||
+ | <tr><td colspan="3"> <h3>Separatory Funnel Extraction of Intracellular Content</h3></td></tr> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | <tr> | ||
+ | <td width="80%" valign="top"> | ||
+ | <p><ol> | ||
+ | |||
+ | <li>Allow cell pellets to remain in -20C freezer for at least one hour, so cells may properly lyse. </li> | ||
+ | <li>Resuspend cell pellet in 5ml 9:1 hexane/acetone solution. Use a micropipette to thoroughly mix cell pellet into hexane/acetone solution. Swirl this solution thoroughly for proper mixing. </li> | ||
+ | <li>Pour this solution into a beaker and add 1.5g sodium sulfate. Swirl this mixture around thoroughly until most of the powder has formed into clumps. | ||
+ | <li>Gently pour the filtered organic phase into a new container without including any of the sodium sulfate. Use a micropipette to transfer any remaining organic phase to the new container, if necessary. </li> | ||
+ | <li>Allow the filtered organic phase to dry under a stream of N2 gas until it reaches <1.5ml, then transfer to a 1.5ml microcentrifuge tube. Allow to continue drying under a stream of N2 gas until it reaches 0.1ml, then transfer to a GC/MS sample bottle. </li> | ||
+ | |||
+ | <tr><td colspan="3"> <h3>Extraction of Organically Soluble Material from Methanococcus – Balch Tube/Sonication</h3></td></tr> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | <tr> | ||
+ | <td width="80%" valign="top"> | ||
+ | <p><ol> | ||
+ | |||
+ | <li>Take & record optical density (OD) values of cultures to be extracted. These cultures should ideally be in the 0.5-0.9 range. </li> | ||
+ | <li>Open the cultures using a de-clamp under the fume hood, relieving the culture of any pressure with a needle, if necessary. </li> | ||
+ | <li>Transfer the culture to a centrifuge-ready container. </li> | ||
+ | <li>Centrifuge the cultures at 11,100G for 20 minutes, or until cell pellet is formed. </li> | ||
+ | <li>After removing the samples from the centrifuge, carefully pour the supernatant into a balch tube, being sure to preserve all of the cell pellet. </li> | ||
+ | <li>Prepare the cell pellet and complete sonication to lyse cells, if planning on completing the extraction for cellular content. </li> | ||
+ | <li> [Either combine the supernatant & cell lysate, or extract them separately.] </li> | ||
+ | <li>Measure out & pour 12-15ml of Methylene Dichloride (DCM) into the balch tube with the extract, there should be a distinct difference between the water phase layer and the organic phase layer. [DCM will be the bottom layer as DCM is denser than water.] </li> | ||
+ | <li>Cap the balch tube, and mix the water phase and organic phase by rapidly vortexing for 60 seconds. Allow any built pressure to escape by briefly removing the cap during mixing. </li> | ||
+ | <li>Allow the water phase and organic phase to settle until a distinct difference is visible again. </li> | ||
+ | <li>Using a micropipette, carefully remove and discard the water phase, leaving only the organic phase in the balch tube. </li> | ||
+ | <li>Pour all of the organic phase into a beaker and add 1.5g of sodium sulfate. Swirl this mixture around thoroughly until most of the powder has formed into small clumps. </li> | ||
+ | <li>Gently pour the filtered organic phase into a new container without including any of the sodium sulfate. Use a micropipette to transfer any remaining organic phase to the new container, if necessary. </li> | ||
+ | <li>Allow the filtered organic phase to dry under a stream of N2 gas until it reaches <1.5ml, then transfer to a 1.5ml microcentrifuge tube. Allow to continue drying under a stream of N2 gas until it reaches 0.1ml, then transfer to a GC/MS sample vial. </li> | ||
+ | |||
+ | <tr><td colspan="3"> <h3>Anaerobic Transformation (1.5ml method - Plating & Enrichment)</h3></td></tr> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | <tr> | ||
+ | <td width="80%" valign="top"> | ||
+ | <p><ol> | ||
+ | |||
+ | <li>Check List of items on Day 1</li> | ||
+ | <ul> | ||
+ | <li> Prepare Minispin & Vortex </li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li> Preheat sand box </li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li> TB and TB+PEG Buffer </li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li> Recipient strain of OD 0.7-1.0 in a rack (always make it at least two days ahead) </li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li> Selection markers </li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li> Media and plates </li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li> Latex gloves, tube locks, tips, pipette (1ml), microcentrifuge tubes </li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li> Plasmid concentration determined (0.4-0.6ug per sample </li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li> 100ml beaker, for dumping supernatant </li> | ||
+ | </ul> | ||
+ | <li>Day 1 (making plates)</li> | ||
+ | <ul> | ||
+ | <li> Melt plates (2 plates per sample) by autoclave, let cool down to 65C </li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li> Add Na2S, antibiotics & additives into plates and let solidify on side way </li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li> Finish check list and prepare chamber (add H2 to 5.0, change catalyst and add CaCl2 </li> | ||
+ | </ul> | ||
+ | <li>Day 2 (transformation and plating)</li> | ||
+ | <ul> | ||
+ | <li> Wear latex gloves, get TWO 1.5ml micro-tubes and place 1.5ml culture (OD 0.7-1.0) into each tube. </li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li> Centrifuge at 3,900 rpm (Minispin) for 10 minutes </li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li> Discard supernatant, add 1ml of TB, vortex (strong: 8th gear) </li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li> Centrifuge at 6,500 rpm for 10 minutes </li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li> Discard supernatant, add 75ul of TB, pipetting up & down for 10 times </li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li> Add 0.2-0.3ug of plasmid and 45ul of TB-PEG, pipetting up & down for 10 times </li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li> Incubate at 37C in sand box for 1 hour </li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li> While waiting, prepare 5ml of broth media plus Na2S in balch tube </li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li> After incubation, combine 2 micro-tubes of cells into 1ml broth (total vol=1.24ml), vortex (strong) </li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li> Take 2 new micro-tubes and add 0.9ml broth to each, now you have ~2ml broth left in balch tube </li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li> Do 10^0, 10^-1 and 10^-2 serial dilution with cells from the previous step, vortex (strong) </li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li> Plate 0.6ml of cells from each dilution, put plates on side way overnight before incubating at 37C </li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li> Innoculate remaining cells (~0.6ml) from 10^0 to the balch tube with 2ml broth, record OD & incubate overnight at 37C </li> | ||
+ | </ul> | ||
+ | <li>Day 3 (enrichment in broth)</li> | ||
+ | <ul> | ||
+ | <li> Record OD again from the final step in Day 2, inoculate 0.5ml to 5ml of selective broth </li> | ||
+ | </ul> | ||
+ | |||
+ | </ol> | ||
+ | </p> | ||
+ | </td> | ||
+ | |||
+ | <td></td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | |||
+ | <tr><td colspan="3"> <h3>Plate Reader</h3></td></tr> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | <tr> | ||
+ | <td width="80%" valign="top"> | ||
+ | <p><ol> | ||
+ | |||
+ | <li>We used the Gen5 Software.</li> | ||
+ | <li>We created a procedure to read both in the spectrum of mCherry and within the auto-flourescence spectrum. </li> | ||
+ | <li>Allow the machine to warm up.</li> | ||
+ | <li>Run a blank plate.</li> | ||
+ | <li>Fill the individual wells of your choosing (96 total) with a maximum of 100uL.</li> | ||
+ | <li>Check all wells that you want to be read in the program.</li> | ||
+ | <li>Run the trail.</li> | ||
+ | |||
Latest revision as of 03:31, 18 October 2014
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PCR | |||||
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Plasmid Extraction | |||||
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Restriction Digestion | |||||
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Gel Extraction | |||||
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Ligation | |||||
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Heat Shock Transformation | |||||
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Formate Media | |||||
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General Salts Solution | |||||
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Oxygen Exposure | |||||
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Components for McM Media | |||||
| Separatory Funnel Extraction of Extracellular Content | ||||
| Separatory Funnel Extraction of Intracellular Content | ||||
| Extraction of Organically Soluble Material from Methanococcus – Balch Tube/Sonication | ||||
| Anaerobic Transformation (1.5ml method - Plating & Enrichment) | ||||
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Plate Reader | |||||
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