Main Page

2021-10-12T20:21:17Z

Alac2:

[[File:PnW.png|thumb|300x300px|Visit our Lab's Webpage at [http://www.phagocytes.ca www.phagocytes.ca]]]
Welcome to the protocol wiki for the lab of [http://phagocytes.ca Dr. Bryan Heit], at the [http://www.uwo.ca University of Western Ontario]. This site contains the protocols, and other information, we frequently use in our lab.  Only laboratory members may edit pages, but anyone is welcome to use these protocols.  If you use these protocol, please cite us.  A link for generating citations can be found on the left side of the page.

 Consult the [http://meta.wikimedia.org/wiki/Help:Contents User's Guide] for information on using the wiki software. 

'''''Lab members:'''''

*To get a wiki account please contact Dr. Heit.
*Log in (upper-right side of screen) to add/edit pages.  
*Please follow [[Editing|these formatting instructions]] when editing/creating protocols.
*To create a new page, [[Create new|follow these instructions]].

 

= Index of Protocols =
{|width="100%"
|- style="vertical-align:top;"
|
==== Lab Operations ====
*[[Entrance Protocol]]
*[[Exit Protocol]]
*[[Labbook Guidelines|Guidelines for Proper Use of Laboratory Notebooks]]
*[[Saving Experimental Files|Proper Saving of Experimental Files]]
*[[Setting Up Network Drives|Setting up Access to Network Drives & Wiki]]
|
==== DNA/Cloning ====

*[[Colony PCR]]
*[[PCR]]
*[[Digests]]
*[[Ligation]]
*[[Gibson Assembly]]
*[[Quick 'n' Easy Competent E. coli|Quick 'n' Easy Competent ''E. coli'' (Dh5a)]]
*[[Competent e coli|Generating Competent ''E. coli'' (TFB, BL21 & ZYCY10P3S2T)]]
*[[E. coli Transduction|Transducing ''E. coli'']]
*[[Transformation]]
*[[Neon® Transfection System]]
*[[Generating Minicircles]]
|
==== Microscopy ====
*[[Competition of Charged Molecules with Lipophilic Cations]]
*[[Immunostaining|Fluorescent Immunostaining]]
*[[Inhibition of Focal Contact Signaling]]
*[[Single Particle Tracking]]
*[[Staining for GSD]]
*[[Reducing Photobleaching]]
*[[Acid Washing Coverslips]]
*[[Live Cell FRET]]
*[[3D Printed PDMS Chambers]]
|
|- style="vertical-align:top;"
|
==== General Protocols ====
*[[Common buffers|Common Buffers]]
*[[Cell Culture Guidelines]]
*[[Bacterial Growth Media]]
*[[Primary Macrophage Culture]]
*[[Freezing and Thawing Cells]]
*[[Raw Cell Transfection]]
*[[J774 Cell Transfection]]
*[[Mammalian Cell Transfection]]
*[[Antibiotics]]
*[[Antibiotic Plates]]
*[[Agarose Gels]]
*[[Water Bath Antibiotic Solution]]
|
==== Protein work ====
*[[Western Blotting]]
*[[Immunoprecipitation]]
*[[Fab preparation]]
*[[Fab Purification by FPLC|Fab purification using FPLC]]
*[[Updated FPLC Size Exclusion Procedure]]
*[[Coomassie Staining]]
*[[Receptor Cross-linking and Activation|Receptor activation by Cross-Linking]]
*[[Stripping & Reprobing Blots|Stripping & Reprobing Blots]]
|
==== Lipids ====
*[[Asymmetric liposomes]]
*[[Lipid Coated Bead Preparation]]
*[[Lipid Extraction from Cells]]
*[[Lipisome and Lipid-Coated Beads]]
|
|- style="vertical-align:top;"
|
==== Phagocytosis Protocols ====

*[[Opsonization]]
*[[Preparation of Silica-Magnetic Beads]]
*[[Synchronised Phagocytosis]]
*[[Phagosome Isolation]]
*[[Primary Human Macrophages]]
*[[Labelled E coli]]
*[[Inside-out Labelling of Bacteria]]
*[[Gentamicin Protection Assay]]
*[[Preparation of Digestion-Tracking Bacteria]]
|
==== Cell Biology ====
*[[Ablation of Recycling Endosomes]]
*[[Cell-Type Specific Transfection Protocols]]
*[[G418 & Puromycin Kill Curves|G418 & Puromycin Kill Curves]]
*[[Apoptosis Detection with AnnexinV and PI]]

|}

= Links to More Protocols =
{|width="100%"
|- style="vertical-align:top;"
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==== General Protocol Sites ====
*[http://www.benchfly.com/ BenchFly] - Free Video Protocols
*[http://www.protocol-online.org/ Protocols Online] - large database of biology protocols
*[http://openwetware.org/wiki/Main_Page Open Wet Ware] - large database of open protocols
*[http://www.molecularstation.com/protocol-links/ Molecular Station] - links to many lab-generated protocols
*[http://www.thelabrat.com/protocols/ TheLabRat] - various protocols & lab resourses.
*[http://www.thelabrat.com/protocols/reagents.shtml Common Buffer Recipes] @ TheLabRat
*[http://www.rsc.org/Publishing/Journals/lc/Chips_and_Tips/index.asp Chips & Tips] - Microfluidics protocols
|
==== Free Science Ebooks ====
*[https://www.gitbook.com/book/petebankhead/imagej-intro/details Analyzing fluorescence microscopy images with ImageJ]
*[http://www.imaging-git.com/applications/bioimage-data-analysis-0 Bioimage Data Analysis] - Free registration required

|}

= Useful Links =
{|width="100%"
|- style="vertical-align:top;"
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==== Molecular Biology Databases ====
*[http://www.ncbi.nlm.nih.gov/gene Pubmed Gene] - Find gene information
*[http://www.ncbi.nlm.nih.gov/refseq/rsg/ Pubmed RefSeq] - Find reference sequences
*[http://www.uniprot.org/ Uniprot] - Find protein sequence & structure
*[http://hapmap.ncbi.nlm.nih.gov/ HapMap] - Find human polymophisms
*[http://www.ncbi.nlm.nih.gov/omim OMIM] - Find human gene-assocations
*[http://www.pantherdb.org/ PANTHER] - Automated Protein Function Classification
*[http://useast.ensembl.org/index.html BioGrid] - Protein Interactions
*[http://useast.ensembl.org/index.html Ensemble Genome] - Browse multiple genomes
*[http://www.proteinatlas.org/ Human Protein Atlas] - Info onn gene exrpession, antibody's, etc
*[http://www.genecards.org/index.shtml Gene Cards] - Condenced information on genes
*[http://www.ihop-net.org/UniPub/iHOP/ iHOP] - Information Hyperlinked Over Proteins
*[http://www.hprd.org/index_html Human Protein Reference Database]
*[http://www.wwpdb.org/ PDB] - Protein Structures
*[http://genetics.bwh.harvard.edu/pph2/ PolyPhen2] - SNP Phenotype Predictor
*[http://sift.jcvi.org/ SIFT] - SNP Phenotype Predictor/db
*[http://www.timetree.org/index.php TimeTree] - evolutionary divergence database
|
==== Molecular Biology Tools ====
*[http://blast.ncbi.nlm.nih.gov/Blast.cgi NCBI Blast]
*[http://ca.expasy.org/ ExPASy Tools]
*[http://www.basic.northwestern.edu/biotools/oligocalc.html OligoCalc] - PCR primer Tm calculator
*[http://tools.neb.com/NEBcutter2/index.php NEB Cutter]
*[http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/isoschizomers.asp NEB Isoschizomers]
*[http://searchlauncher.bcm.tmc.edu/seq-util/Options/revcomp.html Reverse-Complement DNA Sequence]
*[http://www.insilico.uni-duesseldorf.de/Lig_Input.html Ligation Calculator]
*[http://www.addgene.org/ AddGene] - clone by e-mail!
*[http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_pattinprot.html PattenProt] - search genomes for protein patterns
*[http://workbench.sdsc.edu/ Biology Workbench]
*[http://www.ebi.ac.uk/Tools/sequence.html EMBL Sequence Tools]
*[http://www.ncbi.nlm.nih.gov/projects/gorf/ NCBI ORF Finder]
*[http://searchlauncher.bcm.tmc.edu/ BCM Search Launcher]
*[http://swissmodel.expasy.org/ SWISS Model protein modeling]
*[http://searchlauncher.bcm.tmc.edu/seq-search/struc-predict.html Secondary Structure Prediction]
*[http://www.predictprotein.org/ PredictProtein] - Protein structure prediction
*[http://3d-alignment.eu/ STRAP] - Protein aligments with structure
|
===== Microscopy Tools =====
*[http://fbs.robarts.ca/ London Regional Microscopy Facility Bookings]
*[http://www.microscopyu.com/ Microscopy U] - Everything you want to know about microscopes
*[http://jcb.rupress.org/content/166/1/11.full Paper on Image Processing Standards] - How not to loose your job
*[http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.html Fluorophore Spectra Viewer] at Life Technology
*[http://www.mcb.arizona.edu/ipc/fret/ Fluorescent Spectra Database] - FRET and other
*[http://www.mcb.arizona.edu/IPC/spectra_page.htm Yet More Spectra] - from Arizona University
*[http://www.confocal-microscopy.org/ www.confocal-microscopy.org] - Data on LSM methods and equipment
*[http://fiji.sc/wiki/index.php/Fiji FIJI] - FREE imageJ based image processing program
*[http://www.dspguide.com/pdfbook.htm Free] image processing textbook
*[http://www.archive.org/details/Lectures_on_Image_Processing Lectures on image processing]
*[http://vaa3d.org/ VAA3D] FREE viewer for large 3/4/5D datasets
|
|- style="vertical-align:top;"
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==== Journal Resources ====
*[http://www.ncbi.nlm.nih.gov/pubmed/ Pubmed]
*[http://www.ncbi.nlm.nih.gov/pmc/ Pubmed Central (USA)]
*[http://pubmedcentralcanada.ca/ Pubmed Central (Canada)]
*[http://scholar.google.com Google Scholar]
*[http://eigenfactor.org/ Eigenfactor] - free journal imapct scores
|
==== ''In Vivo'' Tools ====
*[http://www.emouseatlas.org/emap/home.html EMAP] - Virtual mouse anatomy
*[http://phenome.jax.org/ Mouse phenome database] - mouse phenotypes 
|
==== Protease Tools ====
*[http://merops.sanger.ac.uk/ MEROPS] - Peptidase database
*[http://www.proteolysis.org/proteases PMAP] - Proteolysis Map
*[http://casbase.org/casvm/index.html CASVM] - Caspace substrate prediction
*[http://bioinf.gen.tcd.ie/casbah/ CASBAH] - Caspase cleaveage site database
|
|- style="vertical-align:top;"
|
==== Genomics Resources ====
*[http://www.gwascentral.org GWAS Central]
|
==== Chemical Tools ====

*[http://www.chemspider.com/ ChemSpider] - General chemistry database
*[http://pubchem.ncbi.nlm.nih.gov/ PubChem] - Chemical structure database
|
==== Lipid Tools ====
*[http://www.lipidmaps.org/ Lipid Maps] - Lipidomics gateway at ''Nature''
*[http://www.avantilipids.com/ Avanti Lipids] - Buy lipids
|}

G418 & Puromycin Kill Curves

2021-03-25T16:19:53Z

Alac2: /* Working Concentrations */

= 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 required is highly dependent on the cell type being transfected. This wiki entry contains both the protocol for performing a kill curve, as well as concentrations we have found to work for various cell lines. 

=== Stock Solutions: ===

*G418 - 50 mg/ml active compound (typically ~70% activity is normal)
**Dilute in PBS, filter and store at 4C
*Puromycin - 10 mg/ml 
**Dilute in water, filter, aliquot in 500 ul or 1 ml stocks and freeze 

=== Kill Curve Protocol: ===

#Search the literature for groups which have used the antibiotic to select the cell line being tested. Use this as the middle concentration and test 10X and 100X less & higher concentrations.
#Plate cells into a 6-well or 12-well plate, let recover at least 12 hours & grow to ~80% confluency. 
#Replace the media, adding the antibiotic at the deisred concentrations
#Every 2 days replace the media, keeping the antibiotic concentration the same*
#The best concentration is the lowest concentration which gives 100% killing at day 3-4 (puromycin) or 7-10 (G418)

*For simplicity it is best to prepare ahead of time tubes of media with each dilution of G418/puromycing and antibiotic/anti-fungal added.

== Working Concentrations ==

{| align="left" cellspacing="1" cellpadding="1" border="1" width="80%"
|-
! scope="col" | Cell Type
! scope="col" | [G418]
! scope="col" | [Puromycin]
|-
| HeLa
| 
| 
|-
| Cho-K1
| 
| 5 ug/ml to 10 ug/ml (1:2000 to 1:1000 dilution)
|-
| RAW 264.7
| 
| 10 ug/ml to 20 ug/ml (1:1000 to 1:500 dilution) 
|-
| J774
| 
| 10 ug/ml to 20 ug/ml (1:1000 to 1:500 dilution) 
|
|-
| Raw 264.7
| 
| 2 ug/ml to 4 ug/ml (1:5000 to 1:2500 dilution) 
|-
| THP-1
| 
| 0.5 ug/ml (1:20,000 dilution) 
|-
|MH-S
|100 ug/ml to 200 ug/ml (1:500 to 1:250 dilution) 
|10 ug/ml to 20 ug/ml (1:1000 to 1:500 dilution) 
|}

J774 Cell Transfection

2021-02-04T19:55:37Z

Alac2: /* Cell Culture */

= Protocol =

===== Cell Culture =====

#Prepare a 12-well plate by adding 1 mL of fresh DMEM per well. If coverslips are required, add one coverslip per well prior to adding media. 
#Wash the cells pre-cultured in a T25 flask with 5 ml of PBS, then remove the PBS. Add 1 mL of trypsin for 5 min at 37oC. Add 4 mL of fresh media to deactivate the trypsin, then determine the cell concentration using a hemocytometer. 
#Add the cells to the 12-well plate according to the following concentration:  
#*500,000 cells/well for subsequent microscopy analysis of transfected cells grown on the coverslips => ~35-40 cells/field-of-view at 63x, with ~20% transfected
#*1 million cells/well for subsequent parachuting of cells onto coverslips
#*Alternatively, if cells are 80-90% confluent, add 3-5 drops of trypsinzed cells into each well
#Rock the plate sideways to evenly distribute the cells.
#Culture the cells overnight at 37oC. 

 

===== Transfection =====

#Thaw the DNA to be used for transfection. 
#Set up a reaction for each well in a microcentrifuge tube:
#*Add 3.3 µg of DNA to 150 µL of serum-free media. Vortex the tube briefly to mix.
#*Add 10 µL of FuGENE-HD Transfection Reagent. Flick the tube 15-20 times to mix. ''  Note:'' The transfection reagent is ethanol-based and evaporates easily. Keep it in the fridge until its addition and do not leave uncapped. Re-wrap in parafilm after use. 
#Let the reaction sit for 15 min at room temperature in the tissue culture hood, allowing the DNA-containing micelles to form. ''  Note'': Do not exceed 20 min of incubation time. 
#Add dropwise the total reaction volume of each microcentrifuge tube to its respective cell well. Rock the plate sideways to mix. 
#Incubate the transfecting cells 24-48 hr at 37oC.

 

===== Paraformaldehyde Cell Fixation =====

#Aspirate the media. Wash the cells twice with non-sterile PBS, at 1 mL/well. '' '' ''Note:'' Be careful not to knock the cells off the coverslips during the wash steps.
#Add 1 mL of 4% PFA (diluted in PBS) into each well. Incubate on the plate shaker for 15 min at room temperature in the dark.
#Remove PFA and wash cells with 1 mL/well of non-sterile PBS for 5 min.
#Cover cells with 1 ml PBS/well and keep plate at 4oC.

Heit Lab Wiki - User contributions [en]

Main Page

G418 & Puromycin Kill Curves

J774 Cell Transfection