Molecular Oncology Laboratory Protocols
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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
A50 How to Submit Samples for DNA Sequencing at UIC Core
A51 How to submit plasmids to plasmidsaurus for full-length vector sequencing
A52 How to use Sanger DNA Sequencing Services by AZENTA
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)
B13 General Protocol for Universal TaqMan qPCR (2024)
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)
C12 How to run RNA "bleach gels"
C13 NucleoZOL RNA Isolation Protocol
C14 MOLab standard RT-PCR reaction using hexamer
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
E1A Cell Culture Vessel Surface Area and Seeding Density
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
E34 Protocol for Cryopreservation of Sf9 and Hi5 Insect Cells
E35 How to flow sort human and mouse adherent cells (updated)
E36 Baculovirus Purification Protocol
F. SKELETAL & STEM CELL BIOLOGY & HISTOLOGY TECHNIQUES
F1 Alkaline Phosphatase Colorimetric Assay (Sigma kit, discontinued)
F2 Alkaline Phosphatase Histochemical Staining
F3 Alizarin Red S Staining of Cells Cultured in Mineralization Medium
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 Chondrocyte Culture
F21A Safranin O Staining Protocol for Cartilage
F21B Modified PAS Staining of Bone-Cartilage Interfaces
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
F46 Trichrome Staining for Bone and Cartilage Tissues
F47 Sample submission form for Histology Core (HTRC)
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
G15 Training and Use of Lago-X Imaging Unit
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