Protocols

Protocols used by the Peccoud Lab: molecular biology, DNA synthesis, DNA sequencing, yeast genetics, and imaging protocols.

gene deletion & gene fusion protocol for yeast

Peccoud Lab Protocols: using PCR to delete a yeast gene or fuse a tag to a gene Introduction Yeast gene deletions or gene fusions (e.g. ORF with epitope tags or FP, promoter replacement, MS2 mRNA tagging) can be generated simply by...

C-terminal tag protocol: Adding a C-terminal tag to a gene

Peccoud Lab Protocol: designing primers to add a C-terminal tag to the (3′) end of a gene via PCR Introduction It is sometimes necessary to generate gene fusion strains with a C-terminal tag or promoter changes by intoducing a PCR amplicon...

N-terminal tag protocol: Adding an N-terminal tag to a gene

Peccoud Lab Protocol: designing primers to add an N-terminal tag to the 5′ end of a gene via PCR Introduction It is sometimes necessary to generate gene fusion strains with an N-terminal tag or promoter changes by introducing a PCR amplicon...

Phenol-chloroform extraction & EtOH precipitation protocol

Peccoud Lab Protocol: purifying and/or concentrating DNA/RNA from various sources by phenol-chloroform extraction and ethanol precipitation. Introduction Sometimes it becomes necessary to concentrate your DNA/RNA or obtain purer DNA/RNA samples. If your DNA is resuspended in nucease-free water, you can...

Primers for gene deletion in yeast (design protocol)

Peccoud Lab Protocol: how to design primers for generating a de novo gene deletion in yeast Introduction Yeast strains carrying single deletions of non-essential genes are commercially available from GE Life Sciences (http://dharmacon.gelifesciences.com/cdnas-and-orfs/non-mammalian-cdnas-and-orfs/yeast/yeast-knockout-collection/). However, these are available only as deletions...

DNA extraction protocol for yeast genomes or plasmids

Peccoud Lab Protocol: Isolating genomic DNA or plasmids from yeast Introduction The following protocol will recover total DNA and, with the indicated modifications, is useful to recover plasmids or genomic DNA. For plasmid recovery, the total DNA is transformed into E....

Coomassie blue staining protocol for protein gels

Peccoud lab Protocol: Staining protein gels with Bio-Safe Coomassie blue Introduction In gel visualization of proteins allows one to determine how evenly loaded the protein sample were, and the general state of the samples – i.e., are the bands sharp...

SDS-PAGE protocol: visualizing proteins on acrylamide gels

Peccoud Lab Protocol: visualizing proteins via sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) Introduction Because cellular extracts contain thousands of different proteins at a wide range of concentrations, it is often difficult to detect and measure specific proteins in these mixes, even when...

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