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Biotechniques Molecular Biology Forum

Molecular biology
PCR - basic method
Reverse transcription (Qiagen Omniscript)
PCR, QPCR and sequencing primers
(Old primer dilution method)
Real-time RT-PCR with SYBR green
Purification of PCR product by ExoSAPit
Purification of short (<200 bp) PCR products - Linear acrylamide
Agarose gel electrophoresis
Agar plate preparation
Trizol protocol for RNA extraction
Genomic DNA prep by DNAzol
DNAzol protocol for DNA extraction
Blood and tissue DNA by Qiagen column
DNA concentration by Yo-Pro fluorescence
RNA and DNA concentration by UV absorbance
Firefly luciferase reagent preparation (homebrew)
Firefly/renilla Promega DualGlo assay protocol in 96 well
DNA extraction from whole blood by salt ppt

Plasmid mutagenesis with DpnI (Stratagene Quikchange)
DNA extraction from FTA cards
Western blotting - UGT1A6
General Western blotting protocol
UGT1A6 exon 1 PCR
UGT1A3'-UTR PCR

UGT real-time PCR primer sets

Cell culture
Selection of cells with Geneticin (G418)
Transient transfection and stable cell lines - HEK293
Freezing and thawing eukaryotic cells - protocol
Lipofectamine 2000 general transfection protocol

Lipofectamine 2000 cotransfection plasmid RNAi

Beta gal assay - Promega
Some good cell culture practices
Caco2 protocol
LS180 protocol

Microsomes, activities, TLC, and HPLC
Liver microsomes preparation
Protein concentration assay by BCA method (Pierce)
Protein (BCA) assay on microtiter plate
UDPGA and NADPH cofactor solutions
UGT activity screen by C14UDPGA-TLC
Development of UGT activity assay by HPLC and preliminary phenotyping
Glucuronidase confirmation of glucuronide metabolite
Deconjugation of drug metabolites in urine
Measurement of soy isoflavones in pet food
HPLC-mass spec buffer preparation
Triazolam in plasma by LCMS

Other methods
Calibration of Ohaus scale
Maintenance of ABI 7300 Real-time PCR machine
QuickCalcs online calculator
Simple Hardy-Weinberg Calculator - Court Lab

 


Comparative and Molecular Pharmacogenomics Laboratory
Department of Veterinary Clinical Sciences,
Washington State University College of Veterinary Medicine

100 Grimes Way, Pullman, WA 99163, USA.
Phone: 509-335-0817; Fax: 509-335-0880
Comments or problems please send email to:
michael.court@vetmed.wsu.edu
Last update: 10 October, 2012

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Comparative and Molecular Pharmacogenomics

Laboratory Protocols

 

 

IMPORTANT - This laboratory has recently relocated to Washington State University in Pullman, WA.