User contributions for Admin

1 February 2021

• 20:1520:15, 1 February 2021 diff hist +82‎ m Main Page ‎No edit summary Tag: Visual edit
• 20:1420:14, 1 February 2021 diff hist 0‎ N File:PnW.png ‎No edit summary current
• 19:5819:58, 1 February 2021 diff hist +4,994‎ N Lipisome and Lipid-Coated Beads ‎ Created page with "= General Notes = *All buffers should be filtered through a 0.2um filter to remove any particulates before use *Avoid plastics until beads/lipisomes are generated, as lipids..."
• 19:5819:58, 1 February 2021 diff hist +962‎ N Lipid Extraction from Cells ‎ Created page with "= Protocol = ===== Initial extraction: ===== #Wash cells 3X with PBS to remove serum. #Remove cells by scraping cells into PBS. #Pellet cells in ultra centrifuge; max spee..." current
• 19:5819:58, 1 February 2021 diff hist +1,429‎ N Lipid Coated Bead Preparation ‎ Created page with "= Protocol = ===== Lipid Preparation: ===== #In a glass container, and using lipids dissolved in chloroform, mix desired lipids to a final quantity of ~4mmol total lipid. #..." current
• 19:5819:58, 1 February 2021 diff hist +4,716‎ N Asymmetric liposomes ‎ Created page with "= Protocol = ===== Outer Bilayer Lipid Preparation:<br> ===== #Measure out the lipid mixtures into sialinized glass vials. Evaporate the chloroform using nitrogen or argon...." current
• 19:5719:57, 1 February 2021 diff hist +10,173‎ N 3D Printed PDMS Chambers ‎ Created page with "== Introduction == Highly detailed imaging chambers can be constructed by 3D printing a mould using DLP (resin) printing, which is then used to cast a PDMS chamber that can be..." current
• 19:5719:57, 1 February 2021 diff hist +6,608‎ N Live Cell FRET ‎ Created page with "Our microscope is capable of FRET measurements using conventional CFP/YFP pairs and their more modern derivatives. We recommend using [https://www.fpbase.org/protein/mturquois..."
• 19:5719:57, 1 February 2021 diff hist +2,244‎ N Acid Washing Coverslips ‎ Created page with "'''WARNING: These procedures involve the use of heated strong acids. Clearly label all containers with warning signs, and wear all appropriate safety equipment.''' Acid clean..."
• 19:5619:56, 1 February 2021 diff hist +8,113‎ N Staining for GSD ‎ Created page with "<u>G</u>round <u>S</u>tate <u>D</u>epletion (GSD) microscopy detects fluorophore positions independently of intensity or staining density.  As such, it is critical that p..."
• 19:5619:56, 1 February 2021 diff hist +3,248‎ N Single Particle Tracking ‎ Created page with "= Notes = *For imaging, use a space ship with the inner surface painted flat black *The dilution for the primary Fab' must be worked out for each antibody, and after each di..." current
• 19:5619:56, 1 February 2021 diff hist +497‎ N Inhibition of Focal Contact Signaling ‎ Created page with "= Protocol = #Cool cells if needed, protocol also works at 37C #Wash 2X with PBS at required temperature #Cover cells with PEG media, incubate 10-20 min #Wash carefully wi..." current
• 19:5619:56, 1 February 2021 diff hist +2,515‎ N Immunostaining ‎ Created page with "This procedure was optimized using the 4G10 (anti-phosphotyrosine) antibody, but should work well for many other antibodies. For a generic protocol, add an additional 20-60min..."
• 19:5619:56, 1 February 2021 diff hist +1,833‎ N Competition of Charged Molecules with Lipophilic Cations ‎ Created page with "= Protocol = #TF cells on coverslips with desired construct, and treat cells as required for the experiment.<br> #Prepare 2x stocks of desired lipophilic compound (see table..." current
• 19:5519:55, 1 February 2021 diff hist +1,541‎ N Apoptosis Detection with AnnexinV and PI ‎ Created page with "==Reagents== *Binding Buffer *AnnexinV-flourophore conjugate (e.g. ThermoFisher AnnexinV-488) *Propridium Iodide ==Protocol== ===Binding Buffer=== Prepare binding buffer, for..." current Tag: Visual edit: Switched
• 19:5519:55, 1 February 2021 diff hist −10‎ m G418 & Puromycin Kill Curves ‎ →‎G418 and Puromycin Kill Curves Tag: Visual edit
• 19:5419:54, 1 February 2021 diff hist +1,948‎ N G418 & Puromycin Kill Curves ‎ Created page with "= G418 and Puromycin Kill Curves = G418 and puromycin can be used to generate stabally transfected cell lines. However, the concentratio of these antibiotics that are require..."
• 19:5419:54, 1 February 2021 diff hist +3,464‎ N Cell-Type Specific Transfection Protocols ‎ Created page with "This page contains the protocol of transfecting specific cell types.  Unless otherwise noted, all transfections are for 12-well (22mm) plates.<br> <br> == RAW 264.7 Mur..." current
• 19:5419:54, 1 February 2021 diff hist +1,350‎ N Ablation of Recycling Endosomes ‎ Created page with "From [http://www.ncbi.nlm.nih.gov/pubmed/15534004 Ang et al. 2004, JCB 167: 531] == <br> Stock solutions == ===== Human Tfn-HRP ===== *0.5 mg/ml in H2O + 20% glycerol ===..." current
• 19:5319:53, 1 February 2021 diff hist +2,380‎ N Stripping & Reprobing Blots ‎ Created page with "It is possible to strip the detection antibodies off of a western blot, and then re-probe that blot with a second set of antibodies.  Every strip will reduce the amount o..." current
• 19:5319:53, 1 February 2021 diff hist +3,786‎ N Receptor Cross-linking and Activation ‎ Created page with "Ligand-independent activation of most receptors can be achieved through antibody-mediated cross-linking.  However, care must be taken, especially on cels of the immune sy..." current
• 19:5319:53, 1 February 2021 diff hist +4,820‎ N Coomassie Staining ‎ Created page with "Thes procedures are adapted from the procedure at [http://www.nationaldiagnostics.com/article_info.php/articles_id/80 National Diagnostics]. = Method 1: Standard (sensitive)..."
• 19:5319:53, 1 February 2021 diff hist +6,325‎ N Updated FPLC Size Exclusion Procedure ‎ Created page with "==Important Notes == * All buffers, 20% ethanol and samples must be pre-filtered using a 0.2 μm filter * Install desired loading loop onto system before initiation, but do..." current
• 19:5319:53, 1 February 2021 diff hist +4,331‎ N Fab Purification by FPLC ‎ Created page with "= Notes<br> = *All buffers must be filtered or otherwise rendered particle-free. *pH 2-10 can be used *Sterile PBS can be used as a buffer. *A maximum of 2ml can be run throu..." current Tag: Visual edit: Switched
• 19:5219:52, 1 February 2021 diff hist +28‎ m Fab preparation ‎ →‎References current Tag: Visual edit
• 19:5219:52, 1 February 2021 diff hist +4,052‎ N Fab preparation ‎ Created page with "== Buffers Required: == <pre>Note: THe volume of buffers prepared is dependent on the starting volume of antibody-containing media. Adjust volumes accordingly. </pre> =====..."
• 19:5119:51, 1 February 2021 diff hist +6,845‎ N Immunoprecipitation ‎ Created page with "== Phosphoprotein Immunoprecipitation == Receptor activation by ligand addition or cross-linking, followed by immunoprecpitation of phosphoproteins, can be used it identify t..." current
• 19:5119:51, 1 February 2021 diff hist +11,652‎ N Western Blotting ‎ Created page with "This is a generic protocol for western blotting.  Optimized procedures for specific blots are listed at the end of this protocol. = Cell Preparation and Lysis<br> = #Pr..." Tag: Visual edit: Switched
• 19:4919:49, 1 February 2021 diff hist −18‎ m Main Page ‎ →‎DNA/Cloning Tag: Visual edit
• 19:4419:44, 1 February 2021 diff hist +2,419‎ N Transformation ‎ Created page with "=== Protocol<br> === #Use either 10-50 ng of DNA in a volume between 10 and 25 μL or entire ligation reaction and place the tube on ice.<br> #Thaw 100 μL of competent ''E...." current
• 19:4419:44, 1 February 2021 diff hist +1,499‎ N Generating Minicircles ‎ Created page with "= Protocol = *Grow an overnight culture of ZYCY10P3S2T containing the appropriate minicircle parental plasmid in 1-5 mL TB + Kan **Recipe for 50 mL Terrific Broth: ***1.2 g Ye..." current Tag: Visual edit: Switched
• 19:4319:43, 1 February 2021 diff hist −2‎ Neon® Transfection System ‎No edit summary current Tag: Visual edit
• 19:4319:43, 1 February 2021 diff hist 0‎ N File:Tip.png ‎No edit summary current
• 19:4319:43, 1 February 2021 diff hist 0‎ N File:Chamber.png ‎No edit summary current
• 19:4219:42, 1 February 2021 diff hist +8,092‎ N Neon® Transfection System ‎ Created page with "The following is a general protocol for using the Neon® Transfection System, along with specifics for optimized use with common cell lines found in the Heit Lab. =Introducti..."
• 19:4219:42, 1 February 2021 diff hist +2,419‎ N E. coli Transduction ‎ Created page with "=== Protocol<br> === #Use either 10-50 ng of DNA in a volume between 10 and 25 μL or entire ligation reaction and place the tube on ice.<br> #Thaw 100 μL of competent ''E...." current
• 19:4219:42, 1 February 2021 diff hist +3,222‎ N Competent e coli ‎ Created page with "= TFB Protocol for DH5a<br> = #Inoculate a single colony from an LB plate into 5ml of LB medium in a plating tube OR inoculate about 1 µL of competent ''E coli ''i..." current
• 19:4119:41, 1 February 2021 diff hist +2,892‎ N Quick 'n' Easy Competent E. coli ‎ Created page with "This protocol can be used to generate competent E. coli of the following strains: *DH5a *DH1 *JM109 This will '''NOT''' work for BL-21 nor ZYCY10P3S2T (minicircle) strains =..." current
• 19:4119:41, 1 February 2021 diff hist +3,053‎ N Gibson Assembly ‎ Created page with "Gibson Assembly, also known as enzymatic or chew-back assembly, is a powerful tool used for DNA cloning. It allows you to manipulate almost any fragment of DNA in any location..."
• 19:4119:41, 1 February 2021 diff hist 0‎ N File:Ga.png ‎No edit summary current
• 19:4019:40, 1 February 2021 diff hist +1,059‎ N Ligation ‎ Created page with "=== Protocol === #Determine the amount of plasmid and insert to be used through the equation X ng plasmid/[(1 M) X bp] = X ng insert/[(X M) X bp]. It is best to have a pasmid..." current
• 19:4019:40, 1 February 2021 diff hist +2,001‎ N Digests ‎ Created page with "=== Protocol === Add the following reagents into a round-lidded PCR tubes:<br> {| cellspacing="1" cellpadding="1" border="1" style="width: 506px; height: 142px;" |- ! scope=..."
• 19:4019:40, 1 February 2021 diff hist +1,198‎ N PCR ‎ Created page with "=== Protocol for Phusion<br> === *Add the following volumes to a round-lidded PCR tube:<br> {| cellspacing="1" cellpadding="1" border="1" style="width: 326px; height: 225px..." current
• 19:4019:40, 1 February 2021 diff hist +726‎ N Colony PCR ‎ Created page with "= Protocol = #Mark a clean agar plate (with required antibiotic) with a grid; make sure top is clearly defined. #Prepare PCR tubes containing 10μl PCR reaction mix/tube. La..." current
• 19:3919:39, 1 February 2021 diff hist +5,723‎ N Gentamicin Protection Assay ‎ Created page with "== Introduction == The gentamycin protection assay is used to monitor the killing or growth of pathogenic bacteria by phagocytes such as macrophages and neutrophils. This assa..." current
• 19:3919:39, 1 February 2021 diff hist +4,912‎ N Inside-out Labelling of Bacteria ‎ Created page with "Many phagocytosis assays require that bacteria be labelled such that internalised bacteria can be clearly delineated from those merely bound to the cell surface. This is achie..." current
• 19:3919:39, 1 February 2021 diff hist +2,221‎ N Labelled E coli ‎ Created page with "This method will produce highly-fluorescent, heat-killed ''E. coli'' for phagocytosis assays. This assay has been optimised for ML35, but should work for other strains of ''E...." current
• 19:3919:39, 1 February 2021 diff hist +3,829‎ N Primary Human Macrophages ‎ Created page with "= Isolation & Culturing Primary Human Macrophages = Human macrophages can be prepared from anticoagulant-treated peripheral blood mononuclear cells.  By culturing mo..." current
• 19:3819:38, 1 February 2021 diff hist +3,801‎ N Phagosome Isolation ‎ Created page with "This isolation is based on the protocol of Lonnbro ''et al<ref name="Pubmed Central">http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2453110&amp;amp;amp;amp;tool..." current
• 19:3819:38, 1 February 2021 diff hist +925‎ N Synchronised Phagocytosis ‎ Created page with "Synchronised phagocytosis is used to ensure that all observed phagocytosis events in a maple have occurred after a similar amount of time.  This is useful for microscopy..." current