General Biochemistry & Molecular Biology

Cloning & Transfection

Cloning

Site-directed mutagenesis

Transfection of HEK293T cells with PEI

  • This is a cheap and effective protocol for transfecting HEK293T cells using PEI.  Note: In Jan 2013 we posted an updated version (version 2) with clarifications requested by users. We have another variant here. We have adapted this protocol for 24 well plates. To see the adapted version click Protocol for 24-Well Plates

 PCR

The PCR resource manual.

  • The how-to guide:qPCR from primer design to testing those primers in preparation for quantitative PCR

cDNA synthesis

  • This protocol is used to synthesis cDNA from isolated RNA using the SuperScript II reverse transcriptase enzyme with the aim to use the synthesized cDNA in subsequent 2-stage RT-qPCR reactions.

Designing primers for qPCR

Two-step qPCR

Two-step reverse transcription quantitative polymerase chain reaction, often abbreviated as 2-Step RT-qPCR or simply as qPCR, is a high-throughput assay design that allows the real-time measurement of the expression level of specific genes at the RNA level. This protocol was written for SYBR green.

Protein

Immunoprecipitation

  • This protocol demonstrates how to immunoprecipitate a protein from a cell lysate.

Co-immunoprecipitation

Western immunoblotting

  • This includes running the gel, transferring to PVDF membrane and preparing antibodies.

 Coommassie blue staining of protein gels

RNA

RNA extraction using TRIzol 

This protocol is used to extract the RNA from tissue or a cell sample using TRIzol and also includes recommendations for 260/280 and 260/230 values when measuring the concentration of extracted RNA using a nanodrop spectrometer.

RNA extraction – lungs

RNA extraction – nasopharynx

RNA Extraction for RNAseq

  • This protocol is for isolating RNA from cell lines or tissues for RNA sequencing.

RNA preparation for RNAseq (RNA sequencing/transcriptomics)

  • The protocol details the steps required fro processing (eukaryote) RNA for RNA sequencing experiments including ribosomal RNA depletion, quality control and quantification, cDNA preparation and labelling with adaptors. 

Other (e.g., bacterial sugars, SDS contamination)

Methylene Green SDS Detection Assay

  •  Is there SDS contamination in your reagent? Quantitate the amount of SDS using this assay based off of the traditional Methylene Blue assay.

Phenol-sulfuric acid Mannose detection assay.

  • This assay is sensitive to mannose from 1 – 100 𝜇g but will also detect other carbohydrates. We use this assay to quantify of complex carbohydrates in the bacterial cell wall