Addgene Protocols

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On this page you'll find practical lab protocols that you can use for a wide range of applications, with videos for select protocols in the right-hand column. You can find even more video content on the Addgene Video Page. We hope that you'll find these protocols useful in your own work. Please feel free to email us at [email protected] with any questions.

Intro to the Lab Bench

Name	Description	(Link opens in a new window) Link to Video
Personal Protective Equipment (PPE) for BSL-1 and BSL-2 Labs	Learn how to best protect yourself when working in BSL-1 and BSL-2 labs	Watch the Video!
Lab Safety for Biosafety Levels One and Two (BSL-1 and BSL-2)	Safety measures for laboratories operating at BSL-1 and BSL-2	Watch the Video!
Water Baths	Proper water bath maintenance and use	Watch the Video!
Pipetting	Learn about selecting the correct pipettor and pipette tip, how to dispense liquids, and how to handle the pipette	Watch the Video!
Centrifugation	Learn about selecting and using a centrifuge to separate different components in a liquid sample
Using a Light Microscope	Learn about the parts of a light microscope and its use
Weighing Reagents	Learn how to weigh laboratory materials on a balance

Basic Molecular Biology

Name	Description	(Link opens in a new window) Link to Video
Making LB Agar Plates	Create plates to culture bacteria in the lab	Watch the Video!
Over-Agar Antibiotic Plating	Quickly add antibiotic to a pre-poured plate	Watch the Video!
Streaking Bacteria	Isolate single bacterial colonies on an agar plate	Watch the Video!
Inoculating a Liquid Bacterial Culture	Prepare and grow bacteria in liquid medium	Watch the Video!
Creating Bacterial Glycerol Stocks	Store bacterial strains or plasmids for long term use	Watch the Video!
DNA Purification	Miniprep, phenol-chloroform extract, and precipitate DNA
DNA Quantification	Measure DNA concentration with a spectrophotometer
Restriction Digests	Cut and analyze plasmid DNA with restriction enzymes
Agarose Gel Electrophoresis	Separate DNA by size on an agarose gel	Watch the Video!
DNA Ligation	Assemble DNA using DNA ligase
Bacterial Transformation	Introduce DNA into a bacterial strain	Watch the Video!
CRISPR Library Amplification	Amplify CRISPR pooled-plasmid libraries
Diagnostic Restriction Digest	Verify a plasmid using restriction enzymes	Watch the Video!
Polymerase Chain Reaction (PCR)	Basic PCR protocol with tips and FAQ	Watch the Video!
Purifying DNA from an Agarose gel	Isolate specific DNA bands cut from a gel	Watch the Video!
How to Design a Primer	Key considerations when designing primers	Watch the Video!
Sequence Analysis	Verify important plasmid features using sequence analysis
RNA Extraction Without a Kit	Extract RNA from tissues or cells without a commercial kit

Plasmid Cloning

Name	Description	(Link opens in a new window) Link to Video
Restriction Cloning	Assemble plasmids by restriction digest and ligation
Cloning by PCR	Generate restriction sites by PCR
Modification by Annealed Oligo Cloning	Add a short stretch of DNA to a plasmid
Gibson Assembly	Combine overlapping DNA fragments in a single reaction
Ligation Independent Cloning (LIC)	Scarless cloning with Type II restriction enzymes and T4 polymerase
pLKO.1 - TRC Cloning Vector	Cloning protocols for using the pLKO.1 vector, a backbone used by the RNAi consortium for targeting human and mouse genes.

Virus

Name	Description	Link to Video
General Transfection	Introduce plasmid DNA to mammalian cells
Lentivirus Production	Produce lentivirus with a polyethyenimine (PEI) transfection protocol
Fluorescence Titering Assay for Lentivirus	Quantify virus using fluorescence measurements
Colony Formation Titering Assay for Lentivirus	Quantify virus by counting antibiotic resistant colonies
Generating Stable Cell Lines with Lentivirus	Genomically integrate your DNA sequence into a cell line
Isolating a Monoclonal Cell Population by Limiting Dilution	Generate monoclonal cell lines from a polyclonal pool of stable cells
AAV Production in HEK293 Cells	Produce adeno-associated virus to deliver your gene of to the target of your choice
AAV Purification by Iodixanol Gradient Ultracentrifugation	Purify adeno-associated virus from a preparation produced in mammalian cells	Watch the Video!
AAV Titration by qPCR Using SYBR Green Technology	Use qPCR to measure the physical titer of your adeno-associated virus preparation	Watch the Video!
AAV Titration by ddPCR	Use ddPCR to measure the physical titer of your adeno-associated virus preparation
Lentivirus Titration by ddPCR	Use ddPCR to measure the physical titer of your lentivirus preparation

Antibodies

Name	Description	Link to Video
Transfection	Introduce plasmid DNA to mammalian cells to produce antibodies
Antibody Purification	Purify recombinant antibodies using Protein A or Protein G columns	Watch the Video!
Coomassie Purity Stain	Determine purity and concentration of recombinant antibodies	Watch the Video!
Western Blot	Separate and detect specific proteins	Watch the Video!
Immunocytochemistry	Use antibodies to detect antigens in cells
Antibody Validation Using the Indirect ELISA Method	Run an indirect ELISA against a purified antigen to demonstrate the expected antibody dose response.