Molecular Oncology Laboratory Protocols

MOLab Biosafety Manual UC Safety UC EHS UC Safety Training IBC Training IACUC Training MOLab Survuval Manual MOLab Law & Order MOLab Cloning Tips MOLab Graduation Checklist Lab Directory Lab Meeting Schedule Lab Jobs Find SBRI J-611 IVIS Bioluminescence Imaging training: video-1 video-2 IVIS Fluorescence Imaging training: video-1 video-2 Amira-Avizo imaging software tutorial Olympus cellSens imaging program manual CorelDraw tutorial: short video long video IACUC Animal bookkeeping log sheets: Tumors Bone/Stem Cells Rats Cell culture hood sign-up sheet Cell culture basics-videos A. BACTERIAL, DNA & CLONING TECHNIQUES A1 Preparation of LB Broth A2 Preparation of LB-Agar A3 Protocol for Pouring LB-Agar Plates A4 Freezing Bacterial Cells for Long Term Storage A5 Preparation of electrocompetent DH10B bacterial cells A6 Preparation of electrocompetent XL-1 Blue bacterial cells A7 Preparation of electrocompetent BJAdEasy1 bacterial cells A8 Preparation of electrocompetent BJ5183 bacterial cells A9 Checking Competency of Electrocompetent Bacterial Cells A10 Transformation of electrocompetent DH10B or XL-1 bacterial cells A11 Transformation of BJAdEasy bacterial cells A12 Transformation of BJ5183 bacterial cells A13 Harvesting Bacteria from Plates A14 Alkaline Lysis Miniprep of Plasmid DNA A15 Large Scale Plasmid Purification (CsCl Banding) A16 Purification of DNA using Wizard plus miniprep kit A17 Bacterial Colony Lifts for Cloning Screening A18 MOLab Standard DNA Cloning/Ligation Protocol A19 T4 DNA Pol blunt-ending or fill-in reactions A20 Protocol for PC8 Extraction of DNA Samples A21 Protocol for Ethanol Precipitation of DNA Samples A22 Isopropanol Precipitation Removing Small Oligomers A23 Preparation of Sephadex G-25 and Sephacryl S-300 A24 Preparing and Using Homemade Spin Columns for DNA Purification from Gels A25 Protocol for large scale restriction digestion reactions A26 SYBR I Green Staining for low amount of DNA A27 Multimerization of Oligonucleotides A28 DNA and Oligo Labeling with T4 kinase A29 DNA Labeling with Random Priming (Oligo labelling) A30 Electrophoretic Mobility Shift Assay (EMSA) A31 DNA Cycle Sequencing Protocol A32 Preparing and Running Genomyx Sequencing Gel A33 Preparation of SSSSS DNA A34 Preparation of Miniprep Plasmid for DNA Sequencing A35 Protocol of Determining DNA, RNA, and Oligo Concentration A36 DNA laddering apoptosis assay A37 Extract DNA From Agarose Gel Using Montage DNA Extraction or Homemade Magic Column A38 How to Download and Analyze DNA Sequencing Files from the Cancer Center A39 Isolation of Bacterial Genomic DNA A40 Protocol for Using Wizard Plus SV Minipreps Kit A41 How to Prepare homemade MB grade Glycogen A42 DNA Gel Extraction using MIDSCI Kit A43 Protocol for Site-Directed Mutagenesis A44 Mouse Tail DNA isolation (updated by Sean Zou) A45 High Speed Plasmid Miniprep using MIDSCI kit A46 New NEB Buffer Chart for commonly used restriction enzymes (by Liang Chen) A47 Protocol for Mag-Bind-mediated RNA Removal for Plasmid A48 How to construct multiplex siRNA vectors using FAMSi system A49 How to prepare antibiotics stock solutions B. PCR TECHNIQUES B1 PCR screening of bacterial colonies B2 Preparatory PCR amplification B3 Protocol for RT-PCR cDNA synthesis (updated by Yang Bi) B4 Manual Touchdown PCR amplification B5 Protocol for Using Gradient PCR on PCRExpress B6 Protocol for Using Touchdown PCR on PCRExpress B7 Standard PCR (From BV’s Cookbook) B8 Purification of Genomic DNA from Paraffin Sections for PCR B9 Real-Time PCR (qPCR) Using OPTICON II (updated by Fang Deng) B10 High fidelity PCR Reaction for Cloning B11 MOLab TqPCR Protocol B12 qPCR technology: A comprehensive guide (Agilent 2012) C. RNA TECHNIQUES C1 Protocol for total RNA isolation using RNAgent kit C2 Preparing and running Northern blotting gel C3 Northern Blotting Gel Transfer and Hybridization C4 Revised Northen Blot protocol-use of Hybridization Oven C5 Protocol for Preparation of Oligo(dT)-Beads C6 Total RNA Isolation Using RNAgent Kit (by Kang) C7 Total RNA preparation Using TRIzol reagent C8 IN SITU HYBRIDIZATION - FROZEN SECTIONS (Polyak Lab) C9 How to Produce and Use the siRNA Cocktails for RNAi Knockdown C10 How to isolate RNA from tissue samples (by Xiaoji Luo) C11 Preparation of RT-PCR for qPCR (by Xiaoji Luo) D. PROTEIN TECHNIQUES D1 Protocol of SDS-PAGE (for 2 minigels) (by Lan Zhou) D2 Protocols for Western Blotting (by Lan Zhou) D3 Protocol for Induction and Purification of GST Fusion Protein (By Lan Zhou) D4 Immunostaining for Paraffin Sections by Hue Luu D5 Protocol of Paraffin Sections for Immunostaining D6 Immunostaining for Paraffin sections D7 Protocol for paraffin sections for immunostaining (by Ou) D8 Immunohistochemical stainning on cultured cells D9 Protocol for Immunostaining of Beta-Catenin in cultured cells D10 BCA Protein Concentration Assay (by Lan Zhou) D11 Coomassie Blue Staining D12 GST Fusion Protein-Large Scale induction and purification (by Kang) D13 GST Fusion Protein-Small Scale Induction and Purification (by Kang) D14 GST-fusion protein Pull-down D15 Homemade SDS-PAGE Gels (by Kang) D16 Western Blotting (Wet Transfer Protocol) (by Kang, updated by Yang Bi) D17 Protocol for Immunoprecipatation D18 How to Use Semidry Transfer Apparatus for Western Blotting D19 Western Blotting (updated by Ying Peng) D20 Immunostaining for Paraffin Sections (Updated by Hue) D21 Chromatin Immunoprecipitation (ChIP) Assay (by Weike Si) D22 BCA Protein Concentration Assay for 96-well Plates (by Yang Bi and Yi Wang) D23 Protein Migration Rate Chat on PAGE (Bio-Rad) D24 Bradford protein assay D25 His-tag purification protocol E. TISSUE & CELL CULTURE TECHNIQUES E1 Procedure for Passage of Mammalian Cells E2 Procedure for transfection of mammalian cells using LipofectAMINE E3 Protocol for Freezing and Thawing Mammalian cells E4 Procedure for establishing stable clones of mammalian cells E5 Making Firefly Luciferase reporters using pBV-Luc E6 Beta-Galactosidase (X-Gal) Staining Assay E7 Protocol for total crystal violet staining of mammalian cells E8 Protocol for preparing complete cell culture medium and G418-containing medium E9 AdEasy Got Easier E10 Hoechst 33258 Staining E11 Prepare Genomic DNA from Mammalian Cell lines (simplified protocol) E12 Hirt protocol for isolating small DNA molecules from mammalian cells E13 Preparation of BMP Conditioned Medium E14 PEI transfection of 293pTP cells for making adenoviruses E15 MTS (Promega) Cell Proliferation Assay E16 Transwell (Boyden Chamber) Cell Migration Assay E17 AdEasy Comprehensive Protocol from CPHG E18 MTT Cell Proliferation Assay E19 Firefly Luciferase (FLuc) Reporter Assay E20 Mouse embryonic fibroblast (MEFs) isolation E21 Quantitative fluorescence measurement using Modulus E22 Drug selection table for commonly used lines (by Chris Song) E23 How to Select Optimal siRNA Target Sites Using SOS-Based Vectors E24 Establishment of Stable Cell Lines Using Pseudotyped Retrovirus E25 Collagen Gel Colony Formation Assay E26 FACS Cell cycle analysis (by Xiaoji Luo) E27 Promega beta-Gal assay E28 Collagen coating of tissue culture dishes E29 Transwell Cell Migration-Invasion Matrigel Assays (by Yuxi Su) E30 How to eliminate Mycoplasma from infected cells using Plasmocin (Invivogen) E31 How to Coat Cell Culture Plasticware with 0.1% Gelatin (Wenli Zhang) E32 Gaussia Luciferase (GLuc) Reporter Assay E33 UC CAT Flow Cytometry Training Videos: Rm S-301 (LSR Fortessa 4-15 HTS) (Quanteon) UC CAT Hands-On Training: NovoExpress Software on the Quanteon & Penteon Flow Basics 2.1: The Basic Staining Protocol Flow Basics 2.2: Optimizing the Basic Cell Staining Protocol Flow Basics 2.3: Panel Design Flow Basics 2.4: Experimental Design and Controls Flow Basics 2.5: Instrument Setup and Automated Compensation F. SKELETAL & STEM CELL BIOLOGY & HISTOLOGY TECHNIQUES F1 Alkaline Phosphatase Colorimetric Assay (Sigma kit, discontinued) F2 Alkaline Phosphatase Histochemical Staining F3 Matrix Mineralization (Alizarin Red S Staining) F4 Matrix Mineralization (von Kossa Staining) F5 How to isolation blood buffy-coat cells by Mike Sun F6 How to isolation blood buffy-coat cells by Boden lab F7 Intramuscular Ectopic Ossification Protocol F8 Hematoxylin & Eosin (H & E) Staining F8-B The Science and Application of H & E Staining by Skip Brown-NWU F9 Hematoxylin & Eosin Staining (Surgical Path Protocol) F10 Protocol for Alcian Blue Staining for Frozen Sections F11 Protocol for Cell and Tissue Fixation F12 Protocol for Isolation and Culture of Articular Chondrocytes F13 Protocol for Micromass Culture (Chondrogenic Activity Assay) F14 In situ Hybridization on Frozen section (Nelly Polyak Lab) F15 In situ Hybridization Protocol (Quan Li Lab) F16 More In Situ Hybridization Protocols F17 Procedure for Micromass Cell Culture F18 Alcian Blue Staining for Micromass Cell Culture F19 Alkaline Phosphatase Assay (BD SEAP Kit) (updated by Ni Tang) F20 Alkaline Phosphatase Assay (WAKO Kit, no longer in use) F21 Safranin O Staining for Micromass Cell Culture F22 Oil Red O Staining of fat cells F23 Growth and Differentiation of 3T3-L1 pre-adipocytes (Kahn lab) F24 Bone Histology Protocols from David Rowe Lab (UConn) F25 How to use Xenogen IVIS 200 Imaging Unit in Carlson ARC F26 ALP Detection by TEM F27 Growing feeder-free ES cells by BayGenomics F28 Isolation of mouse Mesenchymal Stem Cells F29 Safranin-O TypeII Collagen Stains (Lonza) F30 Calcium Deposition Assay (Lonza) F31 Oil Red O Stain (Lonza) F32 Growing and Analysis of hMSCs (Lonza) F33 C2C12 myogenic differentiation protocol F34 PAS Stain of differentiated hepatocytes (by Yang Bi & Ni Tang) F35 ICG Stain of differentiated hepatocytes (by Yang Bi & Ni Tang) F36 IHC Stain of OC and OPN in cultured cells (by Liang Chen) F37 How to genotype KOMP ES-derived mouse tail genomic DNA F38 Organ culture protocol for mouse bone and skeletal development (by Liang Chen) F39 MOLab Protocol for genotyping knockout mice (by Stephannie Kim & Sean Zuo) F40 MicroCT imaging and analysis guidelines (2010 JBMR) F41 MicroCT imaging and analysis (2011 Nature Protocols) F42 Microimaging of bone structure (2009 Nature Reviews Rheumatology) F43 Amira Software tutorial F44 How to use Leica TP1020 Automatic Tissue Processor F45 MOLab Optimized Decalcification Protocol for Decelleularized Bone Matrix G. INSTRUMENT USE & PROCEDURES G1 Detailed protocol for using Kodak Image Station 440CF G2 How to count CPM using the LUMI-SCINT G3 How to use Cyclone Phosphor Imager G4 How to use DU-530 UV Spectrophotometer G5 Procedure for using the inverted Olympus Fluorescence Microscope G6 Protocol of using the LUMI-SCINT for Luciferase Reporter Assay G7 Quick Reference Guide to Use Kodak ImageStation G8 Use of Time Controller for Fluorescence Microscope G9 How to Order Alcohol at U of C G10 How to MAPP A DRIVE G11 Fluorescence Filter Cube Turntable G12 How to Use HP DesignJet 480CA Large Format Printer G13 How to Use PE Victor3 for Luminometry G14 How to Use Cluster3 software H. SOLUTIONS, BUFFERS & MISCELLANEOUS H1 Commonly Used Buffers and Solutions H2 Homemade Agarose Tubes for Desalting and Dialysis H3 Preparation of BLOTTO-10 H4 CAGE Protocol H5 BV Lab Survival Manual H6 Alcohol Order and Delivery SOP at U of C H7 How to Get Access to Carlson Animal Use and Care Facility
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