McManus Lab Protocols


General Cell Biology
24-well Plate Spin Infection
Culturing Immortalized Dicer Floxed MEFs
DNA Isolation from ES Cells -96 well plate
Electroporation of targeting construct
Freezing 96-well master plates
Freezing ES cells
Growing/splitting E14 ES cells
Lac Z staining mouse embryos
Passaging/Splitting 96-well Master Plate into Three Plates
Picking ES cell colonies and transferring them to 96-well plates
Rubidium Chloride Competent Cell Protocol
Staining for beta-galactosidase activity
Thawing ES cells

General Molecular Biology
8-cell Embryo Collection
A PCR-based strategy for cloning short hairpin sequences: “PCR shagging"
Actin Cre genotyping
Chemical Synthesis of ImpA (adenosine 5'-phosphorimidazolide)
CIP
Cloning via Restriction Digest
LacZ Genotyping
Making Long dsRNA from In Vitro Transcriptions
Metabolic Labeling Using 32P
MicroRNA and siRNA Cloning Protocol
P32 Probe Preparation
Preparing 96-well Genotyping Plate for genomic DNA Extraction
Site-Directed Mutagenesis (Stratagene protocol)

Virus
CLONING SMALL HAIRPINS INTO LENTIVIRAL VECTORS
LentiLox Vector Design
Lentiviral Harvest and Concentration
Lentiviral Transfection
Lentiviral Transfection Protocol for a 10cm dish
Lentiviral Vectors Perivitelline Injection
Lentivirus harvest and concentration

microRNA
Library cloning protocol - mir30 context
miRNA microarrays
Small RNA Northern Blotting

RNAi
Library cloning protocol - shRNA context
Lipid siRNA transfection
Lymphocyte siRNA electroporation transfection
PCR template method for dsRNA Synthesis
siRNA transfection with HiPerfect (MIN6 cells)
NIH3T3 transfection with Jetprime Polyplus