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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).

• 15:36, 22 July 2024 User account Akelm talk contribs was created
• 16:18, 25 June 2024 Jcomstock talk contribs created page Electroporation and Plating of M. xanthus transformants (Created page with "Dialysis Prior to electroporation of the plasmid into M. xanthus, excess salts from the plasmid extraction need to be removed, as salt will interfere with the current that passes through the sample. You will float a cellulose membrane (pore size 0.025 µm) in a well containing water, allowing for the exchange of water and salt but not plasmid. 1. Fill a 60mm Petri dish 2/3 full with sterile, diH2O. You'll need one per plasmid. Before you add the membranes, ensure that...")
• 11:23, 19 June 2024 EricMiller talk contribs created page Working with GitHub (Created page with "GitHub is a way to keep track of your code for a project, while also allowing a team to work independently on the same project. It is a bit unintuitive to use; it combines using a web browser with command line functions (through the Mac terminal, or Windows Command Line). =Installing Git (one time only)= Macs: Install Xcode, which will include Git automatically <br> <code> xcode-select --install </code> Windows: https://git-scm.com/download/win To check to se...")
• 09:48, 30 May 2024 User account Ariadnekelm talk contribs was created
• 09:36, 30 May 2024 User account CVeras talk contribs was created
• 13:49, 15 May 2024 Mtyoung talk contribs created page File:U SMUR.png
• 13:49, 15 May 2024 Mtyoung talk contribs uploaded File:U SMUR.png
• 13:44, 15 May 2024 Mtyoung talk contribs created page File:V SMUR.png
• 13:44, 15 May 2024 Mtyoung talk contribs uploaded File:V SMUR.png
• 13:43, 15 May 2024 Mtyoung talk contribs created page File:Metals SMUR.png
• 13:43, 15 May 2024 Mtyoung talk contribs uploaded File:Metals SMUR.png
• 13:41, 15 May 2024 Mtyoung talk contribs created page File:BM SMUR.png
• 13:41, 15 May 2024 Mtyoung talk contribs uploaded File:BM SMUR.png
• 13:32, 15 May 2024 Mtyoung talk contribs created page Streptococcus mutans Transformation Media Recipe (SMUR) (Created page with "S. mutans Recombination (SMUR) Media Recipe:")
• 13:54, 25 February 2024 Jcomstock talk contribs created page Spore Assay (Created page with "1. Harvest overnight M. xanthus culture during log growth phase, wash twice in TPM, and concentrate to %x10^9 cells/ml. You will likely need several mL of culture to plate out a spore assay. Each large TPM plate will need 4 groups of 5 20uL spots (400uL per plate, where one plate = 1 replicate). You may wish to use the 15mL conical tubes and the centrifuge in superlab to prepare cells. 2. While you are washing and preparing cells, leave TPM plates open to dry by flame o...")
• 10:44, 22 February 2024 User account Jinu Ryu talk contribs was created
• 18:20, 31 January 2024 Jcomstock talk contribs created page Updated Liquid Biological Waste Disposal Protocol (BSL1) (Created page with "Liquids considered biological waste are those that contain living cells or DNA, including bacterial cultures (and supernatants from those cultures). These must be disposed of according to the biohazard waste stream detailed below and cannot just be poured down the sink. If you have questions, ask Jess! For overnight bacterial cultures in flasks, remove what you need for experiments and leave the remaining culture in the flask covered with foil. Place in the plastic bin...")
• 12:42, 8 January 2024 EricMiller talk contribs created page Measuring Burst Size (Created page with "# Prepare 10mL of media and set up cells to incubate for 1 hour in the 37C shaking incubator #* Set up for 10^8 cells which is 12.5 ul for Black WT aliquots found in the -80 freezer # After 1 hour, add phage for a total amount of 10^6 phage #* For P21 of the pre-adapted phage, the amount needed is 8.62 ul # Start the timer as soon as the phage has been added and incubate at 37C #* The best way to do this now is to place in the shaking incubator after adding phage and whe...")
• 13:14, 25 December 2023 User account Pottslewis82 talk contribs was created
• 16:23, 27 October 2023 Jcomstock talk contribs created page Propidium Iodide Staining on Agar Plates (Created page with "This protocol is for staining fruiting bodies on agar/agarose plates with propidium iodide (PI). 1. Put on gloves to prevent the PI stain from contacting your skin. 2. Remove an aliquot of PI stain (one of the PCR tubes of purple PI stain) from the -20C freezer. 3. Once thawed, dilute the PI stain 1:100 in TPM (2uL into 198uL for example). You need 20ul of diluted PI solution per spot that you want to stain, so make sure to calculate enough. 4. Mix the PI stain t...")
• 15:11, 26 October 2023 User account Jnoppenber talk contribs was created
• 15:09, 26 October 2023 Jcomstock talk contribs created page Heat Fixing and Staining (Created page with "1. From an overnight liquid culture, pipet 2-5uL of sample into the center of a microscope slide. Spread across the middle third of the plate and allow to dry. 2. Heat fix once dry by passing the slide through the flame 2-3 times quickly--do not keep over the flame too long. 3. OVER THE SINK, apply a few drops of crystal violet or safranin stain so that the cells are covered. Wait 30 seconds, and rinse gently with water. Blot dry with a paper towel. 4. Image with t...")
• 15:54, 20 October 2023 User account Kim.palacios talk contribs was created
• 14:40, 9 October 2023 User account Kpalacios talk contribs was created
• 13:46, 17 September 2023 EricMiller talk contribs uploaded a new version of File:MEE Floor Plan Upper Section Final.png
• 12:15, 17 September 2023 EricMiller talk contribs created page Media and Passaging (Created page with "==Minimal Media Recipe== {| class="wikitable" style="margin:auto" |+ LeMaster-Richards Minimal Media (LeMaster and Richards, 1982) |- ! Final Volume !! Water !! 4x Minimal Salts !! 2M MgSO4 !! Trace Minerals (4000x) !! Glucose (if needed) !! |- |10ml || 7.5ml || 2.5ml || 10ul || 2.5ul || xxul |- |100ml || 75ml || 25ml || 100ul || 25ul || xxul |- |250ml || 180ml || 62.5ml || 250ul || 62.5ul || xxul |} *Glycerol should be added to a final concentration of xx% (or xx%...")
• 11:50, 17 September 2023 EricMiller talk contribs created page Plaque Assays with Soft Agar (Created page with "==Preparing Soft Agar== The soft agar should be prepared first, likely at least an hour before you pour # Make sure the water bath has enough water and is warming to 42C # You will use about 4 mL of soft agar per plate, calculate total volume of agar needed accordingly * Always prep at least one extra plate # Loosen the cap of the soft agar bottle and microwave the bottle of agar until it is fully liquid: * Check for bubbling about every 15 seconds * Y...")
• 20:49, 14 September 2023 User account Scrooke talk contribs was created
• 13:49, 14 September 2023 EricMiller talk contribs created page Streptococcus DNA Extraction - Genome Prep (Created page with "== Reagents Needed== * 50mM Tris-10mM EDTA, pH 8 Autoclaved. **Note: max concentration that will dissolve EDTA (without altering pH) is 0.1M. Making 10mL of the 50mM Tris Buffer Solution + 10mM of the EDTA will result in a 10% diluted Tris Buffer solution. We are working to troubleshoot this problem for the next time around. **If working with small volumes such as 10mL that need to be sterilized, an alternative to autoclaving is filter sterilization. Use either a 10 or...")
• 20:16, 12 September 2023 User account Ahspence talk contribs was created
• 17:05, 11 September 2023 User account BuzzBzBuzz talk contribs was created
• 17:02, 8 September 2023 User account Emmaalmo talk contribs was created
• 13:50, 22 August 2023 EricMiller talk contribs uploaded File:C+Y SpeumoCompetence.png
• 13:50, 22 August 2023 EricMiller talk contribs created page File:C+Y SpeumoCompetence.png
• 13:21, 10 August 2023 EricMiller talk contribs created page SOE PCR (Splicing by Overlap-Extension) (Created page with "Eventually, a 50ul PCR. * 5x GC buffer (or whichever PCR buffer used): 10ul * Templates: Up to 100ng of largest fragment ** Equivalent molarity for all other fragments * dNTPs: 1ul * Water:To 47.5ul * Phusion: 0.5ul * PCR for 15 cycles "using the annealing temperature of the homologous regions". * Extension time: use the length of the (n-1) longest fragments, where n is the total number of fragments * Immediately after these 15 cycles: ** Add 1ul each of outside prime...")
• 12:41, 24 July 2023 EricMiller talk contribs created page Streptococcus mutans Transformation (Created page with "Day 1: Grow up a single colony of S. mutans (from BHI plate in fridge or incubator) in 3ml of of BHI, in 37 degree 5% CO2 incubator. Day 2: #Warm two tubes of BHI to 37 degrees; one with 3ml (for bacteria growth), and one with ~7ml (as a blank and extra in case the OD600 of the experimental one needs to be diluted down) in either the incubator or heat block. #Turn on Spectrometer #1:20 Dilution of overnight culture into the Exp BHI (from step 1) (150uL of the overnight...")
• 12:28, 23 June 2023 Dnguyen talk contribs created page Prepping a Submerged Culture (Created page with "1. Obtain 60 mm petri plates and overnight culture, and wipe down the bench with 70% ethanol. Make sure that you are setting up nearby the incubator. 2. Turn on the spectrophotometer, and while waiting, label the lid of the petri plate with initials, strain, date, and any treatments. 3. Measure the absorbance of your overnight culture using the spectrophotometer, and calculate the density according to the protocol "Measure Absorbance of M. Xanthus Culture" 4. In a con...")
• 10:18, 15 June 2023 Jcomstock talk contribs created page Colony PCR to confirm correct insert (Created page with "1. Obtain 3 PCR tubes per sample, and two extra for controls. Label the tubes on the top and sides as follows: one ‘u’ for the untransformed E. coli control, one “-” for the negative control with no E. coli cells. The rest you will label in the next step. 2. Choose three patches of transformed E. coli per sample that display good growth on the LB/kan plate. Record in your notebook which patches you have chosen from, and label the rest of the PCR tubes with th...")
• 10:47, 12 June 2023 Jcomstock talk contribs created page Image Analysis in Fiji (Created page with "Saving Time Series and a Time-lapse Move 1. File>Import>Image Sequence... 2. Import image sequence as a virtual stack. 3. File>Save As...>AVI to save as a movie file that is readable across devices. Here you have lots of options. Consider the frame rate that you would like (this depends on the number of images you have). Typically I compress so that 1 second represents 1 hr of real time. This would be 60fps if you took one image every minute, and 30fps if you took 1 im...")
• 16:03, 2 June 2023 Jcomstock talk contribs created page Generating Frozen Stocks of Strains (Created page with "1. Obtain an overnight culture of M. xanthus during log growth phase. Label a cryovial (2mL) on the top with your strain name, and on the side with strain name, date, your initials, and any information such as resistance markers and/or fluorescence. 2. Spin down 1.5ml of culture in a microfuge tube at max speed for 1 min and pipet off supernatant. 3. To the same tube, add another 1.5mL of culture, spin at max speed for 1 min, and pipet off supernatant. 4. Resuspend...")
• 10:20, 2 June 2023 Jcomstock talk contribs created page M. xanthus genomic DNA extraction with Zymo (yellow) kit (Created page with "1. Turn a heat block to 55C. 2. If using an overnight culture, spin down 1.5mL of cells in a microfuge tube. If using cells from a plate, scrape up some culture with a p1000 pipet tip and resuspend it in 500uL of TPM, PBS, or DNA elution buffer from the kit. Spin these cells down at max speed for 1 min to create a pellet. 3. Pour and pipet of supernatant, and resuspend pellet THOROUGHLY in 200uL of TPM, PBS, or DNA elution buffer. 4. Place tube in the freezer for 5...")
• 09:30, 26 May 2023 Jcomstock talk contribs created page Generate St Curve for OD600 to Cells/mL conversion (Created page with "1. Obtain O/N M. xanthus cultures at different densities (ideally relatively equally spaced to achieve close to 0.2, 0.4, 0.6, and 0.8 OD600. 2. Measure the absorbance of these samples and convert values to OD600 by factoring in the path length. Record these values. 3. Melt down CTTSA in the microwave, using caution not to boil it over. Heat in increments of 30 seconds once it starts melting. 4. Aliquot 4mL of CTTSA into as many tubes as is needed for your experime...")
• 15:49, 25 May 2023 Tommy talk contribs created page Plate Colonies Using CTTSA (Created page with "1. Warm up plates you’re using to 32°C. 2. Warm up a heat block to 55°C and place sterile test tubes into it. Have an equal number of test tubes to plates you’re pouring. 3. Determine how many cells you want to plate to determine the volume of cells you add to the CTTSA later. If you want to be able to count colonies, usually try to have ~200 - ~500 cells be added to the CTTSA. 4. Melt CTTSA in the microwave. This usually takes 3-4 minutes, depending on the amount...")
• 12:06, 25 May 2023 Jcomstock talk contribs created page Development Assay on Agarose Plates (Created page with "1. Measure the density of an overnight M. xanthus culture using the spectrophotometer. Before spinning and prepping your samples, calculate the volume of TPM starvation buffer that you will use to bring your overnight culture to a concentration of 5x10^9 cells/mL that we typically use for starvation assays. If you take 1mL of your overnight culture at the density that you calculated, you can use the C1V1=C2V2 calculation. (1mL)(O/N culture cells/mL) = (5x10^9 cells/m...")
• 11:46, 25 May 2023 Jcomstock talk contribs created page Measure Absorbance of M. xanthus Culture (Created page with "1. Turn on the spectrophotometer so it can warm up. 2. Obtain one 13mm glass test tube for each sample that you want to measure density for, plus one to blank. 3. Add 3mL of uncultured CTTYE growth media to the tube that will serve as your blank. 4. Add 2.5mL of uncultured CTTYE to labeled sample test tubes, and add 0.5mL of bacterial culture to the correspondingly labeled tube. **Note, if your sample is particularly dilute, add 1mL culture and 2mL nutrient media...")
• 11:12, 22 May 2023 User account Mlangdon talk contribs was created
• 11:11, 22 May 2023 User account Dnguyen talk contribs was created
• 21:37, 11 May 2023 Dcapcha talk contribs created page Western Blot (Created page with "==Goal== * To extract protein samples from the bacterial host, run them through an SDS-PAGE gel, and use antibodies to detect specific proteins in the size-separated protein samples. This technique is useful for confirming the presence of a particular protein inside or outside of the bacterial host. ==Protocol== ===Day 1=== ====Induction and Overnight Incubation of Bacterial Culture==== '''Day 2: Obtaining Supernatant and Lysate Samples from Bacterial Culture''' '''Da...")
• 10:03, 31 March 2023 Jcomstock talk contribs created page EcoRI digest of plasmid (Created page with "Since the TOPO pcr2.1 plasmid has EcoRI cut sites on either side of the PCR insert, you can perform a restriction digest with EcoRI enzyme to check the size of the insert after plasmid extraction. This ensures that the plasmid you extracted from the E. coli liquid culture is consistent with your colony PCR results. (Remember that your culture could become contaminated with something else between colony PCR and plasmid extraction, so this digest serves as a verification b...")
• 09:28, 31 March 2023 Jcomstock talk contribs created page Plasmid Isolation with BioBasic Miniprep Kit (Created page with "1. On the day before your minipreps you will use sterile technique near a Bunsen burner to set up 3ml of LB culture with 50ug/ml of kanamycin (or other appropriate antibiotic) (add 5ul of the 50mg/ml stock solution) for each colony of interest. Using a sterile P200 pipette tip touch one colony from your patch plate and then drop it into one culture tube. Be careful to record which culture corresponds to which patch on your patch plate. Place cultures (with lids at the lo...")