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19:54, 1 February 2021 Admin talk contribs created page 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...")
19:54, 1 February 2021 Admin talk contribs created page 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. == RAW 264.7 Mur...")
19:54, 1 February 2021 Admin talk contribs created page Ablation of Recycling Endosomes (Created page with "From [http://www.ncbi.nlm.nih.gov/pubmed/15534004 Ang et al. 2004, JCB 167: 531] == Stock solutions == ===== Human Tfn-HRP ===== *0.5 mg/ml in H2O + 20% glycerol ===...")
19:53, 1 February 2021 Admin talk contribs created page 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...")
19:53, 1 February 2021 Admin talk contribs created page 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...")
19:53, 1 February 2021 Admin talk contribs created page 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)...")
19:53, 1 February 2021 Admin talk contribs created page 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...")
19:53, 1 February 2021 Admin talk contribs created page Fab Purification by FPLC (Created page with "= Notes = *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...") Tag: Visual edit: Switched
19:52, 1 February 2021 Admin talk contribs created page 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> =====...")
19:51, 1 February 2021 Admin talk contribs created page 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...")
19:51, 1 February 2021 Admin talk contribs created page 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 = #Pr...") Tag: Visual edit: Switched
19:44, 1 February 2021 Admin talk contribs created page Transformation (Created page with "=== Protocol === #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. #Thaw 100 μL of competent ''E....")
19:44, 1 February 2021 Admin talk contribs created page 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...") Tag: Visual edit: Switched
19:43, 1 February 2021 Admin talk contribs created page File:Tip.png
19:43, 1 February 2021 Admin talk contribs uploaded File:Tip.png
19:43, 1 February 2021 Admin talk contribs created page File:Chamber.png
19:43, 1 February 2021 Admin talk contribs uploaded File:Chamber.png
19:42, 1 February 2021 Admin talk contribs created page 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...")
19:42, 1 February 2021 Admin talk contribs created page E. coli Transduction (Created page with "=== Protocol === #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. #Thaw 100 μL of competent ''E....")
19:42, 1 February 2021 Admin talk contribs created page Competent e coli (Created page with "= TFB Protocol for DH5a = #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...")
19:41, 1 February 2021 Admin talk contribs created page 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 =...")
19:41, 1 February 2021 Admin talk contribs created page 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...")
19:41, 1 February 2021 Admin talk contribs created page File:Ga.png
19:41, 1 February 2021 Admin talk contribs uploaded File:Ga.png
19:40, 1 February 2021 Admin talk contribs created page 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...")
19:40, 1 February 2021 Admin talk contribs created page Digests (Created page with "=== Protocol === Add the following reagents into a round-lidded PCR tubes: {| cellspacing="1" cellpadding="1" border="1" style="width: 506px; height: 142px;" |- ! scope=...")
19:40, 1 February 2021 Admin talk contribs created page PCR (Created page with "=== Protocol for Phusion === *Add the following volumes to a round-lidded PCR tube: {| cellspacing="1" cellpadding="1" border="1" style="width: 326px; height: 225px...")
19:40, 1 February 2021 Admin talk contribs created page 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...")
19:39, 1 February 2021 Admin talk contribs created page 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...")
19:39, 1 February 2021 Admin talk contribs created page 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...")
19:39, 1 February 2021 Admin talk contribs created page 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....")
19:39, 1 February 2021 Admin talk contribs created page 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...")
19:38, 1 February 2021 Admin talk contribs created page 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;tool...")
19:38, 1 February 2021 Admin talk contribs created page 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...")
19:38, 1 February 2021 Admin talk contribs created page Preparation of Silica-Magnetic Beads (Created page with "Silica (SiO2) beads and silica-coated magnetic beads are a substrate for the attachments of a broad range of biomolecules through polar interactions. Lipids,...")
19:38, 1 February 2021 Admin talk contribs created page Opsonization (Created page with "Opsonization allows phagocytes to efficiently recognize particles, by coating them with opsonins such as IgG isotype antibodies. This page contains the protocols for opsonisin...")
19:37, 1 February 2021 Admin talk contribs created page Water Bath Antibiotic Solution (Created page with "= Reagents Needed = *Copper Sulfate (Cu2SO4) *EDTA *Dish soap or other detergent (i.e. Tween/Triton) = Preparation = Fill water bath with tap water (typ...")
19:37, 1 February 2021 Admin talk contribs created page Agarose Gels (Created page with "==== Protocol ==== ===== For a 0.7% gel: ===== #Set up gel-casting dock. #Add 0.14 g of agarose to a 250mL Erlenmeyer flask. #Add 400 µL of 50X TAE and 20 ml of d...")
19:37, 1 February 2021 Admin talk contribs created page Antibiotic Plates (Created page with "=== Protocol === #Add 20 g of LB agar to a 1 L flash. #Fill flask to the 500 mL line with dH2O. #Autoclave the flask. Note: have a water bath set to 50°C. #Whe...")
19:37, 1 February 2021 Admin talk contribs created page Antibiotics (Created page with "== Protocol == ==== Ampicillin ==== Preparation of 5 mL of 100 mg/mL: #Add 0.5 g of ampicilin into a 12 mL tube. #Add 2.5 mL of ddH2O into the tube and mix g...")
19:36, 1 February 2021 Admin talk contribs created page J774 Cell Transfection (Created page with "= Protocol = ===== Cell Culture ===== #Prepare a 6-well plate by adding 2 ml of J774 Cell media per well. If coverslips are required, add one coverslip/well prior to add...")
19:36, 1 February 2021 Admin talk contribs created page Raw Cell Transfection (Created page with "= Protocol = ===== Cell Culture ===== #Prepare a 6-well plate by adding 2 ml of Raw Cell media per well. If coverslips are required, add one coverslip/well prior to addi...")
19:26, 1 February 2021 Admin talk contribs created page Freezing and Thawing Cells (Created page with "= Freezing Mammalian Cells = #In a T75 flask, grow cells to 80-90% confluency, or 75-80% density for suspended cells. #Collect cells (trypsinize/scrape adherent cells, etc)....")
19:25, 1 February 2021 Admin talk contribs created page Primary Macrophage Culture (Created page with "= Isolation and Culture of Mouse Bone Marrow-Derived Macrophages = === Protocol === ===== Materials ===== *Dulbecco's minimal essential medium (DMEM) from Invitroge...")
19:25, 1 February 2021 Admin talk contribs created page Bacterial Growth Media (Created page with "=== LB Broth === To 200 mL ddH2O add: *Pre-made: **6.25g Miller LB powder *'''''OR''''' **2.5g Tryptone **2.5g NaCl **1.25g Yeast extract...")
19:25, 1 February 2021 Admin talk contribs created page Cell Culture Guidelines (Created page with "= Maintenance of Common Cell Lines = {| width="95%" border="1" cellpadding="1" cellspacing="1" |- | '''Cell Type''' | '''Media''' | '''Maintenance''' | '''Splitting''' | '''N...")
19:25, 1 February 2021 Admin talk contribs created page Common buffers (Created page with "= Cell Culture = ===== 1x PBS ===== To 800ml ddH2O add: *8g NaCl *0.2g KCl *1.44g Na2HPO4 (sodium phosphate dibasic) ...") Tag: Visual edit: Switched
19:23, 1 February 2021 Admin talk contribs created page Setting Up Network Drives (Created page with "The laboratory has a server on which all work-related files must be stored. Before you can access these folders you must get a user ID and password from Dr. Heit. Please...")
19:23, 1 February 2021 Admin talk contribs created page Saving Experimental Files (Created page with "= Logging Computer Files = It is not uncommon for experiments to generate large quantities of computer files containing raw data, analyzed data, statistics, intermediary stag...")
19:23, 1 February 2021 Admin talk contribs created page Labbook Guidelines (Created page with "= Preamble = Your laboratory notebook is an important tool that goes well beyond research management and can have important implications for issues ranging from intellect...")

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