C. Elegans Genetic Protocols

C. elegans EMS Mutagenesis http://cobweb.dartmouth.edu/cgi-bin/cgiwrap/~ambros/protocol.cgi?id=26

Knockout Protocol

Genomic DNA prep

Integrating extrachromasomal arrays-- http://cobweb.dartmouth.edu/cgi-bin/cgiwrap/~ambros/protocol.cgi?id=4

Integration via Gamma Radiation -- http://www.biochem.northwestern.edu/ibis/morimoto/research/Protocols/IX.%20C.%20elegans/E.%20Genetics/1.%20Integration%20via%20gamma.pdf

MMS integration

Joe’s mRNA Prep -- http://www.k-state.edu/hermanlab/protocols/joes_mrna_prep.htm

F2 Mutant Screen - non "clonal" -- http://cobweb.dartmouth.edu/cgi-bin/cgiwrap/~ambros/protocol.cgi?id=27

C. elegans RNA Prep Protocol

Expression Lab - RNAi Protocol

Cosuppression in C. elegans – Ketting, et al. pdb ...

EMS mutagenesis

A Guide to Genetic Mapping in C. elegans-- http://uwacadweb.uwyo.edu/UWmolecbio/

Introduction of Double-Stranded RNA in C. elegans by Injection ...

Introduction of Double-Stranded RNA in C. elegans by Feeding ...

Introduction of Double-Stranded RNA in C. elegans by Soaking ...

Isolation of RNA from C. elegans -- Ketting et al. pdb ...

Mapping C. elegans Mutants using STS Polymorphisms -- http://cobweb.dartmouth.edu/cgi-bin/cgiwrap/~ambros/protocol.cgi?id=32

Preparation of C. Elegans Genomic DNA --C

Procedure: --Single Worm PCR

C. elegans gene knockout protocol - new

EMS Mutagenesis Of C. Elegans

Model Organisms/C. Elegans Protocols

C.elegans Gene Knockout Protocol by TMP/UV Method

Nature Protocols: RNAi screens in Caenorhabditis elegans in a 96 ...

Caenorhabditis briggsae methods *

TOTAL RNA-ISOLATION FROM C. ELEGANS ...

RNAi in C. elegans.pdb ...

Gene Knockout Protocol by TMP/UV Method

Identification of 1088 New Transposon Insertions of Caenorhabditis ...

Springer Protocols: Abstract: Intracellular Ca2+ Imaging in C. elegans

C. Elegans methods Floatation, hypochlorite treatment; liquid Culture, RNA Prep ...

Genes misregulated in C. elegans deficient in Dicer, RDE-4, or RDE .

Online Text for “Improving the Caenorhabditis elegans Genome Annotation ...

Homologous gene targeting in Caenorhabditis elegans by biolistic ...

Cloning and characterization of the C.elegans histidyl- tRNA ...

Isolating Mutants of the Nematode Caenorhabditis elegans That Are ...

MultiSite Gateway Recombination System Using a C. Elegans ...

Methods - C. elegans - Cenix Protocol for Production of large Scale dsRNA ...

Towards a mutation in every gene in Caenorhabditis elegans ...

Mos as a tool for genome-wide insertional mutagenesis in C. Elegans ...

IngentaConnect Shotgun cloning of transposon insertions in the Genome of C. Elegans ...

Preparation of C. Elegans Genome DNA - C

Genetics and Neurobiology of C. elegans - Publications

Shotgun cloning of transposon insertions in the genome of C. Elegans...

Identification of 1088 New Transposon Insertions of Caenorhabditis Elegans ...

Genome-wide RNAi screening in Caenorhabditis elegans

Biolistic transformation in C. elegans using unc-119 rescue

Discovery of novel alternatively Spliced C. Elegans Transcripts by Computational Analysis of Sage Data...

Using Stress Resistance to Isolate Novel Longevity Mutations in ...

Biomedical Pathways

BIOCHEMICAL PATHWAY DIAGRAMS

Stages of Catabolism

Calvin Cycle Flow Diagram

Glycolysis

Glycolysis Enzyme Mechanisms

Glyoxylate Cycle

Aerobic Glucose Metabolism

Fatty Acid Oxidation Reactions

Glycolysis/Gluconeogenesis Pathways

Fatty Acid Synthase Complex

Gluconeogenesis in the Liver

Fatty Acid Biosynthesis Reactions

Pentose Phosphate Pathway

Pyruvate-Malate Cycle

Glycogen Cascade Control

Fatty Acid Regulation

Pyruvate DH Complex Reactions

Kreb's Urea Cycle

Kreb's TCA Cycle

Basic Folate Metabolism Pathways

Kreb's TCA Cycle Intermediate Metabolism

Purine Biosynthesis

Malate-Aspartate Shuttle

Purine Degradation

(Sourece: Richard A. Paselk, Humboldt State University)

Other Resources of Biochemical Pathway

Metabolic pathway - Wikipedia, the free encyclopedia

In biochemistry, a metabolic pathway is a series of chemical reactions occurring within a cell. In each pathway, a principal chemical is modified by ...
en.wikipedia.org/wiki/Metabolic_pathway

Image results for biochemical pathways

ExPASy - Biochemical Pathways and Pathway Map

Digitized version of wall charts courtesy Boehringer Mannheim et al, divided into Metabolic Pathways and Cellular and Molecular Processes, maintained by the ...
www.expasy.ch/cgi-bin/show_thumbnails.pl

Pathway Tools Query Page

Each dataset contains classification hierarchies for pathways, for reactions ( the enzyme nomenclature system), for compounds, and for genes. ...
pathway.yeastgenome.org/biocyc/

Pathways

Pathways is provided an an ancillary to General, Organic and Biochemistry and Concepts of Biochemistry. Please send any comments or suggestions to Jim Hardy ...
ull.chemistry.uakron.edu/Pathways/index.html

Yeast Biochemical Pathways

Yeast Biochemical Pathways are created using the Pathway tools software developed by Peter Karp and his colleagues at SRI International. ...
www.yeastgenome.org/biocyc/

T cell purification from splenocytes

Procedure:

Remove the spleen.

Grind the spleen with the flat end of a syringe in 5 ml of RPMI on a 100 mm culture dish.

Pass through all cells through a cell strainer into a 50 ml tube. Wash the cell culture dish with 5 ml of RPMI twice and combine all cells.

Spin down the cells and remove the supernatant. Re-suspend the cell pellet into 5 ml of red blood cell lysis buffer followed by incubation at RT for 2 min. Dilute cells with 45 ml of RPMI or 1X PBS.

Spin down cells and re-suspend the cell pellet in 1 ml of RPMI/10% FCS. Incubate the cells at RT for 30 min.

Purify T cells with a sterile Nylon column. Wet the column with 5 ml of RPMI / 10% FCS followed by another 5 ml wash. Leave 1 ml of medium on the top of the column.

Incubate the column at 37oC for ~ 1 hour.

Open stop cock and allow media to run through column. Add 2 ml media and allow this to run through the column. Add 1 ml of splenocytes (1-2 x 108 cells). Allow cells to enter column. Add another 1 ml of media and allow to enter column. Cover the column with 1 ml media and incubate at 37oC for one hour.

Collect the T cells by washing column with 10 ml of media. Do not plunge, as this removes B cells.

Spin and re-suspend the cells and count them.

To 1 ml of cells (1-6 x107) add 8 µl (8 µg) of anti-MHCII and / or 8 µl (4 µg) of anti-CD8.

Incubate on ice for 30 min. Add 9 ml of PBS or RPMI. Spin and re-suspend the cells into 1 ml of complement. Incubate for 30 min at 37oC. Commercially available complement reagent is prepared by re-suspending the lyophilyzed pellet in 1 ml of cold H2O. This should be diluted in 9 ml of RPMI.

After incubation, add 9 ml of RPMI or PBS. Repeat antibody-complement cycle once more. Wash the cells with RPMI and count.

Other Protocols:

B Cell Isolation

Dynal Biotech (Invitrogen) - Dynabeads ( T Cell Isolation Procedure)

Fractionation of T and B Cells Using Magnetic Beads Protocol –

Isolation of Ly-1+/CD5+ B Cells by Cell Sorting Protocol –

Isolation of Resting B Lymphocytes from Sixteen Mouse Spleens Protocol –

Isolation of Resting B Lymphocytes Protocol –

Lymphocyte Isolation and Culture Protocol –

MagCellect Mouse CD3 T Cell Isolation Kit

Nature Protocols: Generation of multivirus-specific CTLs

Protocol: Immunofluorescence / confocal microscopy : B cells and T Cells.

R&D Systems - MagCellect* Mouse CD3+ T Cell Isolation ( manual)

The Immune System - in More Detail

Aseptic Technique

• Protect eyes, mucous membranes, open cuts and wounds from contact with biohazard material

• Use gloves, goggles, mask, and protective clothing as necessary.

• Carry out all culturing operation in a laminar flow hood.

• Disinfect all surfaces prior to use with a disinfectant solution.

• Swab down the working surface liberally with 70% ethanol.

• Periodically spread a solution of 70% ethanol over the exterior of gloves to minimize contamination. Replace them if torn.

• In case of any spill, spread a solution of 70% alcohol and swab immediately with non-linting wipes.

• Discard gloves after use and do not wear them when entering any other lab area. Bring into the work area only those items needed for a particular procedure.

• Leave a wide clear space in the center of the hood (not just the front edge) to work on. Do not clutter the area to prevent blockage of proper air flow and to minimize turbulence.

• Swab with 70% alcohol all glassware(medium bottles, beakers, etc.)before placing them inside the hood.

• Arrange the work area to have easy access to all of it without having to reach over one item to get at another (especially over an open bottle or flask).

• Use sterile wrapped pipettes and discard them after use into a biohazard waste container.

• Check that the wrapping of the sterile pipette is not broken or damaged.

• Inspect the vessels to be used:

a. T-flask – Must be free from visible contamination or breakage, or lack container

identification. The plastic covering the flask must be intact.

b. Bottles – Check for cracks, expiration dates.

c. Spinners flasks – Check for cracks, expiration dates, and proper assembly.

Discard any biohazard or contaminated material immediately.

Never perform mouth pipetting. Pipettor must be used.

When handling sterile containers with caps or lids, place the cap on its side if it must be laid on the work surface.

Make sure not to touch the tip of the pipette to the rim of any flask or sterile bottle.

Clean the work area when finished by wiping with 70% alcohol.

(Source: Openwetware)

Angiogenic Models-chorioallantoic membrane assay

Biomacromolecules - CAM preparation

CAM preparation. IPW research units. Prof. D. Neri · Prof. H. Wunderli - Allenspach · Prof. U. Spichiger - Keller · Prof. H.P. Merkle · Prof. U. Quitterer ...
www.pharma.ethz.ch/institute_groups/biomacromolecules/protocols/cam

Chorioallantoic Membrane Vascular Assay (CAMVA)

Chorioallantoic Membrane Vascular Assay (CAMVA). Theory: Vascular damage (i.e. ghost vessels, capillary injection, or. hemorrhaging) of the chorioallantoic ...
www.iivs.org/pages/methods/CAMVAsummarysheet.pdf

The gelatin sponge–chorioallantoic membrane assay

The gelatin sponge–chorioallantoic membrane assay. Here we present a method for the quantification of angiogenesis and antiangiogenesis in the chick embryo ...
www.natureprotocols.com/2006/06/23/the_gelatin_spongechorioallant.php

THE CHICK CHORIOALLANTOIC MEMBRANE AS A MODEL TISSUE FOR SURGICAL Retinal Research Simulation.
factors in ocular tissues of normal rabbits on chorioallantoic. membrane assay. Tohoku J Exp Med 1988;154:63–70. 10. Prost M. Experimental studies on the ...
www.stanford.edu/~palanker/publications/CAM%20as%20retinal%20model.pdf

Chorioallantoic membrane assay: vascularized 3-dim.

PubMed is a service of the US National Library of Medicine that includes over 16 million citations from MEDLINE and other life science journals for ...
www.ncbi.nlm.nih.gov/pubmed/11547121

Assessment of Angiogenic Factors The Chick Chorioallantoic Membrane Assay.

Assessment of Angiogenic Factors The Chick Chorioallantoic Membrane Assay. By: Adam Jones3, Chisato Fujiyama3, Stephen Hague3, Roy Bicknell4 ...
www.springerprotocols.com/Abstract/doi/10.1385/1-59259-137-X:119

Chicken chorioallantoic membrane assay (Cytokines & Cells ...

Chicken chorioallantoic membrane assay. The following COPE entries contain this entry term or one of its hypertext synonyms: ...
www.copewithcytokines.de/cope.cgi?key=Chicken%20chorioallantoic%20membrane%20assay

THE SHELL-LESS CHIC CAM ASSAY: A MODEL FOR STUDYING MELANOMA

ANGIOTROPISM AND EXTRAVASCULAR MIGRATORY METASTASIS
http://www.med.miami.edu/mnbws/documents/06Lugassy.pdf

Chorioallantoic Membrane Vascular Assay - Alternative Ocular Irritation Testing

Chorioallantoic Membrane Vascular Assay Alternative Ocular Irritation. Day 7 - Vascular development of the Chorioallantoic Membrane. ASSAY HIGHLIGHTS: ...
www.camva.com/

The gelatin sponge-chorioallantoic membrane assay.

The gelatin sponge–chorioallantoic membrane assay ... quantification of angiogenesis and antiangiogenesis in the chick embryo chorioallantoic membrane (CAM) ...
www.nature.com/nprot/journal/v1/n1/abs/nprot.2006.13.html

angiogenic models

Classical angiogenesis assays include the chick chorioallantoic membrane, rabbit cornea assay, sponge implant models, matrigel plugs and conventional tumor ...
www.med.unibs.it/~airc/sandra/models.html

Evaluating Compounds Affecting Angiogenesis.indd

ASSAY. In a typical angiogenesis evaluation using the CAM assay,. fertilized chick eggs are incubated at 37°C and specific. humidity (60%) for 3 to 4 days. ...
www.sri.com/biosciences/pdf/EvaluatingCompoundsAffectingAngiogenesis.pdf

All Mouse Models Federated Database, NIH

34 Records Found

Note: Please click on the Mouse Name for more information

Note: Please click on the Mouse Name for more information
Mouse Name	Gene Name		Mutation Type	Manipulation Type
Aprt null	adenine phosphoribosyl transferase [APRT]		Null	targeted
ATM null	ataxia telangiectasia mutated (includes complementation groups A, C and D) [ATM]		Null	targeted
Brca1 cre Null	breast cancer 1, early onset [BRCA1]		loxP Sites	targeted
creK5			None selected	transgenic
CycD1bK5(A870G/exon5)	cyclin D1 (PRAD1: parathyroid adenomatosis 1) [CCND1/Cyclin D1]		None selected	transgenic
Cyclin D1b tg	cyclin D1 (PRAD1: parathyroid adenomatosis 1) [CCND1/Cyclin D1]		None selected	transgenic
Cyclin Db1 (A870G/exon 5)	cyclin D1 (PRAD1: parathyroid adenomatosis 1) [CCND1/Cyclin D1]		None selected	transgenic
DNAjC3(tm-gfp)	DnaJ (Hsp40) homolog, subfamily C, member 3 [DNAJC3]		Null	targeted
Exo 1 null	exonuclease 1 [EXO1]		Null	targeted
Fen1 null	flap structure-specific endonuclease 1 [FEN1]		Null	targeted
Gal4 E2F1	E2F transcription factor 1 [E2F1]		None selected	transgenic
Ku70 null	Thyroid autoantigen 70kDa, Ku antigen [G22P1]		Null	targeted
MBD4 null	methyl-CpG binding domain protein 4 [MBD4]		Null	targeted
MCAT Tg	Mitochondrial - Targeted Catalase [MCAT]		None selected	transgenic
MIh1 null	mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli) [MLH1]		Null	targeted
Msh2 (G674A) ATPase	mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli) [MSH2]		Null	targeted
Msh2 null	mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli) [MSH2]		Null	targeted
Msh6(T1217D)	mutS homolog 6 (E. coli) [MSH6]		None selected	targeted
P53(R270H)	tumor protein p53 (Li-Fraumeni syndrome) [TP53]		Point Mutant	targeted
P53.R270H/balancer-Cre	tumor protein p53 (Li-Fraumeni syndrome) [TP53]		Point Mutant	targeted
P53.R270H/K14Cre	tumor protein p53 (Li-Fraumeni syndrome) [TP53]		Point Mutant	targeted
P53.R270H/Wap-Cre	tumor protein p53 (Li-Fraumeni syndrome) [TP53]		Point Mutant	targeted
P53(S389A)	tumor protein p53 (Li-Fraumeni syndrome) [TP53]		Point Mutant	targeted
PCNA Pol130-104	proliferating cell nuclear antigen [PCNA]		None selected	targeted
Plap-Fs			Null	transgenic
Plap-ti			Null	transgenic
POLdelta1 (D400A)	polymerase (DNA directed), delta 1, catalytic subunit [POLD1]		Point Mutant	targeted
PRKR	protein kinase, interferon-inducible double stranded RNA dependent [PRKR]		Point Mutant	transgenic
pUR 288-lacZ 30			Null	targeted
pUR 288-lacZ 60			Null	targeted
WRN K577M Tg	Werner syndrome [WRN]		Point Mutant	transgenic
			Point Mutant	transgenic
			Point Mutant	transgenic
XPV null		polymerase (DNA directed), eta [POLH]	Null	targeted