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Combined display of all available logs of Microbial Ecology and Evolution Lab Wiki. You can narrow down the view by selecting a log type, the username (case-sensitive), or the affected page (also case-sensitive).
- 11:53, 29 March 2023 User account Tommy talk contribs was created
- 10:26, 24 March 2023 Jcomstock talk contribs created page Ligation of PCR product into TOPO 2.1 vector (Created page with "Before proceeding with ligation, please make sure that you have done a PCR clean-up reaction if necessary, following the Zymo DNA clean and concentrator kit. If needed [only if PCR product has been sitting in the freezer for >5 days]: Adding A’s To ensure the presence of 3’ A overhangs on the PCR product add 1 to 5 units of Taq polymerase (1ul of GoTaq) to your PCR tube. Place in PCR machine programmed to run 94oC for 3min and incubate the tube at 72°C for 10 mi...")
- 11:58, 22 March 2023 Jcomstock talk contribs created page Media Protocols (Created page with "CTTYE Media (For 1L) 1. Start with 700mL of water in a 2L flask with a stir bar. If not making plates, you can mix right in the graduated cylinder. 2. Add 10mL of Tris (pH 8.0), 10 mL of 0.8M MgSO4, 1 mL of 1M KPO4 (pH 7.6). 3. Mix in 10g of casitone (sometimes labeled as Casein Peptone), 2 g of yeast extract. 4. Transfer to graduated cylinder and add water to a final volume of 1L. 5. Transfer back to flask to remove stir bar. 5. Add 15g agar (if making plates). If mak...")
- 10:33, 21 March 2023 Jcomstock talk contribs created page PCR Amplification from Genome (Created page with "1. Extract template M. xanthus DNA with genomic DNA miniprep kit or other protocol. Dilute 1:10 to use for this PCR protocol (you will receive already diluted DNA). Note: check freezer first we probably have some isolated gDNA. 2. Prepare PCR reaction master mix. Calculate for the number of reactions plus 1. You will run your experimentalsamples w ith M. xanthus template DNA and your designed primers, a positive control with M. xanthus template DNA and 16s rRNA primer...")
- 10:18, 21 March 2023 Jcomstock talk contribs created page Rehydrating New Primers (Created page with "Follow this protocol to rehydrate lyophilized primers. 1. Place all primer vials into the centrifuge and spin at ~13k rpm for 1 min to make sure the primer is at the bottom of the tube and will not float out as you open the vial. 2. Check the label of the vial. It should contain the amount of oligo in nanomoles. To make a 100mM stock solution of your primer, add 10x the amount of nanomoles of primer in uL. So if you have 18.5 nmol of primer, add 185uL of PCR water to...")
- 14:22, 9 March 2023 Xiwei Huang talk contribs created page Notes and Troubleshooting (Created page with "Notes and Troubleshooting: ● If the plaques (lawn clearings) are not visible: ○ Potentially adjust amount of phage added ○ Ask for help and consider substituting soft agar for an even lower concentration ● Titer- The concentration of infective phages within a stock ○ Titer is calculated in PFU/mL ○ PFU- Plaque Forming Units/ mL ● Remember that for a viral plaque assays, we are looking for “clear spots”, not colonies ● Overnight incubati...")
- 14:19, 9 March 2023 Xiwei Huang talk contribs created page Calculating Virus Titre (Created page with "1. Remove plates from incubator 2. Plaques formed should look like cleared out circles on the lawn 3. Each plaque is the result of one phage lysis one host bacterium, with the progeny from this lysis killing the surrounding bacteria 4. Determine which dilutions have about 70-300 plaques each 5. Count the number of plaques and write them down a. Use the ipad ticker and a sharpie for fast counting 6. After counting, average the amount between replicates for each dil...")
- 14:15, 9 March 2023 Xiwei Huang talk contribs created page Pouring the Plaques (Created page with "The top layer will consist of an equally sized layer made of the same broth and soft agar (.8%) mixed with 30 uL of bacterial culture and 100 uL of phage dilution 1. To prepare for pouring the phage layer: a. Prepare all phage pipettes b. Prepare filtered tips for phage c. Check that your dilutions are represent and organized d. Check that you collected all your warmed plates from the incubator e. Aliquot 4 mL of soft agar into your warming glass tubes f. Place b...")
- 14:07, 9 March 2023 Xiwei Huang talk contribs created page Serial Dilutions of Phage (Created page with "1. Label 8, microcentrifuge tubes with 10^-1, 10^-2,10^-3 all the way down to 10^-8 a. These will serve as our dilution tubes 2. Add 900 uL of LB broth to each tube 3. Vortex the phage stock. 4. To the first tube (10^-1), add 100 uL of phage stock and vortex 5. Change the pippette tip. 6. To the second tube (10^-2), take 100 uL of tube 1, add it to the next dilution, and vortex the second tube 5. Repeat step 5 and 6 down to the 10^-8 tube a. Double check yo...")
- 13:59, 9 March 2023 Xiwei Huang talk contribs created page Preparing the Plates (Created page with "1. Prepare and label your petri dishes a. Dish should have: Date, Your name, Strain, Dilution, and Duplicate ID b. Try to label with small handwriting around the edges to make future plaque counting easier c. The dilution labels on the petri dishes should only correspond to the serial dilutions that you wish to plate (for example: 10-6 -> 10- 8, so 3 plates), do not plate every serial dilution d. If you are working in multiples (like triplicate...")
- 13:48, 9 March 2023 Xiwei Huang talk contribs created page Preparing the Soft Agar (Created page with "1. The soft agar should be prepared first, likely at least an hour before you pour a. Make sure the water bath has enough water and is warming to 42C b. You will use about 4 mL of soft agar per plate, calculate total volume of agar needed accordingly i. Always prep at least one extra plate c. Loosen the cap of the soft agar bottle and microwave the bottle of agar until it is fully liquid: i. Check for bubbling about every 15 seconds ii.You will kno...")
- 08:11, 17 February 2023 Jcomstock talk contribs created page Making a Broth Culture from an Agar Plate (Created page with "Making a broth culture from an agar plate To do essentially all experiments with M. xanthus, we need to make overnight broth cultures in CTTYE. This allows us to control the cell density more carefully for phenotype assays. 1. Obtain a sterile flask with an overturned sterile beaker as a lid (cabinet under the incubators). It is crucial that you use a sterile flask for this. If one is not available, talk to Jess and we can figure out a solution. 2. Working by the stat...")
- 07:59, 17 February 2023 Jcomstock talk contribs created page Culture Cells from a Frozen Stock (Created page with "Culture Cells from a Frozen Stock We store all M. xanthus strains as frozen stocks in the -80C freezer. Each week you will need to make a new nutrient agar plate of cells that we can use for research. Note: do not attempt to make broth cultures straight from frozen stock as the risk for contamination is very high. 1. Remove the necessary CTTYE plates from the 4C fridge. Let it come to room temperature, or if pressed for time, place in the 37C incubator for 5 min. 2....")
- 11:07, 15 February 2023 User account Jcomstock talk contribs was created
- 11:06, 15 February 2023 User account Smohammed talk contribs was created
- 13:32, 30 January 2023 EricMiller talk contribs created page Fixing Cells for Microscope/Flow Cytometry Work (Created page with "===Fixing Cells for Microscope/Flow Cytometry Work=== ==Goal== *Goal: To fix cells (kill and preserve cell structures) so that they can be viewed using a microscope or a flow cytometer. ==Pre-Protocol questions== First, answer these questions: * Log phase growth is best for viewing cells; are your cells actively growing? * If these cells have a fluorescent protein in their genome, are they actively making it? Do they need to be in log growth phase for expres...")
- 13:14, 30 January 2023 EricMiller talk contribs created page Detailed Lab Task Descriptions (Created page with "Detailed Lab Task Descriptions === Serological Pipetes === === Media === === Medical Waste === === Balance/Microwave/Gel Box === === Tips/Tubes === === Glassware === === Virkon === === Water/Ethanol === === Pipettes ===")
- 14:40, 27 January 2023 EricMiller talk contribs created page Virkon (Created page with "* Clean up any Virkon containers that are older than overnight, using the protocol below. * If a flask of Virkon is not yellow (for example, if it is clear), add 1 spoon of Virkon and wait at least 1 hour (or overnight) before cleaning. * Ensure that the strainer is over the sink drain so that no pipets accidentally go down the drain. * Remove green inoculating loops and cotton swabs; put into medical waste. * Strain the Virkon contents through the pasta strainer. * Pic...")
- 14:34, 27 January 2023 EricMiller talk contribs created page Pipettes (Created page with "* On Friday, wipe down all pipettes in lab with 70% ethanol on Friday. Use a paper towel sprayed with 70% ethanol; do not spray directly onto the pipettes. * Lastly, and right before leaving the lab for the day, do the same for the phage pipettes last to prevent any possible contamination. It is key to do this last, to not inadvertently contaminate the lab with phage. If you are concerned about this, feel free to not do this portion of the task.")
- 14:30, 27 January 2023 EricMiller talk contribs created page Balance/Microwave/Gel Box (Created page with "* Ensure that the balance, inside of the microwave, and DNA/protein gel box areas are clean and tidy once during the week. * If needed, spray a paper towel with 70% ethanol and wipe clean. * This includes the salt residue around the gel area. * Dried agar inside of the microwave might require a quick scrub to clean, using the sponge near the sink area.")
- 14:30, 27 January 2023 EricMiller talk contribs created page Glassware (Created page with "* Wash any glassware labeled 'Waste' that are returned by Nicole — use only tap water and the bottle brush if needed, and give a final quick rinse with Milliq water. * Put away clean, dry glassware that are on the drying racks. Keep the sink area tidy. * Washed, dry green test tube caps go below the 5%, 37 degree incubator, in a used, yellow Virkon container. * Re-fill soap as needed from large bottle under sink. * If we have less than 1.5 racks of sterile test...")
- 12:58, 27 January 2023 EricMiller talk contribs created page Balance/Microwave/Gel Box/Virkon (Created page with "* Ensure that the balance, inside of the microwave, and DNA/protein gel box areas are clean and tidy once during the week. * If needed, spray a paper towel with 70% ethanol and wipe clean. * This includes the salt residue around the gel area. * Dried agar inside of the microwave might require a quick scrub to clean, using the sponge near the sink area.")
- 12:56, 27 January 2023 EricMiller talk contribs created page Water/Ethanol (Created page with "* If either of the Milliq water tanks by the sink are below 1/3 full, fill them up with the Milliq water machine in the Fairman lab. Use the black cart (either in the lab or across the hall, in the equipment room with the large -80 freezers) to transport the tanks. You will need Eric, Jess, or someone currently in the Fairman lab to let you in; therefore, this is really a 'business hours only' task. ** Caution: one of the tanks by the sink is instead 70% ethanol; do not...")
- 12:53, 27 January 2023 EricMiller talk contribs created page Glassware (no 50 mL flasks) (Created page with "* Wash any glassware labeled 'Waste' that are returned by Nicole — use only tap water and the bottle brush if needed, and give a final quick rinse with Milliq water. * Put away clean, dry glassware that are on the drying racks. Keep the sink area tidy. * Washed, dry green test tube caps go below the 5%, 37 degree incubator, in a used, yellow Virkon container. * Re-fill soap as needed from large bottle under sink. * If we have less than 1.5 racks of sterile test...")
- 15:56, 24 January 2023 User account Xiwei Huang talk contribs was created
- 13:53, 22 January 2023 User account Mtyoung talk contribs was created
- 22:56, 16 December 2022 Dcapcha talk contribs created page Running SDS-PAGE Gels (Created page with "==Goal== *Goal: To run an SDS-PAGE gel with the end goal of visualizing the protein on the gel ==Pre-Protocol Questions== # Do you need to know the amount of protein (ug) to load, or does it not matter? If you need to know, you must run a Bradford on your protein samples # Do you know if you have enough loading buffer, running buffer, and gel stain (Coomassie, silver, etc.)? # Are you planning to do a Western blot with this SDS-PAGE gel? This protocol is strictly for vi...")
- 22:25, 16 December 2022 User account Dcapcha talk contribs was created
- 13:51, 16 December 2022 Ekhgn0 talk contribs created page File:Screen Shot 2022-08-31 at 9.56.38 PM.png
- 13:51, 16 December 2022 Ekhgn0 talk contribs uploaded File:Screen Shot 2022-08-31 at 9.56.38 PM.png
- 13:50, 16 December 2022 Ekhgn0 talk contribs created page File:Protein Purification Buffer Components.png
- 13:50, 16 December 2022 Ekhgn0 talk contribs uploaded File:Protein Purification Buffer Components.png
- 13:50, 16 December 2022 Ekhgn0 talk contribs created page File:Protein Affinity Column on Clamp Stand.jpg
- 13:50, 16 December 2022 Ekhgn0 talk contribs uploaded File:Protein Affinity Column on Clamp Stand.jpg
- 13:50, 16 December 2022 Ekhgn0 talk contribs created page File:Figure 1 - Amicon® Ultra-4 device.png
- 13:50, 16 December 2022 Ekhgn0 talk contribs uploaded File:Figure 1 - Amicon® Ultra-4 device.png
- 13:49, 16 December 2022 Ekhgn0 talk contribs created page File:Desalitnation Process - User Guide Amicon Ultra-4 Centrifugal Filter Devices.png
- 13:49, 16 December 2022 Ekhgn0 talk contribs uploaded File:Desalitnation Process - User Guide Amicon Ultra-4 Centrifugal Filter Devices.png
- 13:49, 16 December 2022 Ekhgn0 talk contribs created page File:Table 2 and 3 - User Guide Amicon Ultra-4 Centrifugal Filter Devices.png
- 13:49, 16 December 2022 Ekhgn0 talk contribs uploaded File:Table 2 and 3 - User Guide Amicon Ultra-4 Centrifugal Filter Devices.png
- 13:49, 16 December 2022 Ekhgn0 talk contribs created page File:Table 4 - User Guide.png
- 13:49, 16 December 2022 Ekhgn0 talk contribs uploaded File:Table 4 - User Guide.png
- 13:47, 16 December 2022 Ekhgn0 talk contribs created page File:MEE Floor Plan Upper Section Final.png
- 13:47, 16 December 2022 Ekhgn0 talk contribs uploaded File:MEE Floor Plan Upper Section Final.png
- 13:46, 16 December 2022 Ekhgn0 talk contribs uploaded File:MEE Floor Plan Lower Section NoDrawerLabels 1.png
- 13:46, 16 December 2022 Ekhgn0 talk contribs created page File:MEE Floor Plan Lower Section NoDrawerLabels 1.png
- 13:46, 16 December 2022 Ekhgn0 talk contribs uploaded File:S olei.png
- 13:46, 16 December 2022 Ekhgn0 talk contribs created page File:S olei.png
- 13:46, 16 December 2022 Ekhgn0 talk contribs uploaded File:PCR Troubleshooting.png
- 13:46, 16 December 2022 Ekhgn0 talk contribs created page File:PCR Troubleshooting.png