Protocols

/Protocols

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

DNA Fragment Analysis via 4200 Agilent TapeStation

Materials Consumables: D5000/Genomic Sample Buffer D5000/Genomic Ladder Strip Tubes/96 well plate Strip Caps/Aluminum Foil Seal D5000/Genomic Screentape Hardware: 4200 TapeStation Agilent Computer Vortexer Prepping Samples Launch the '4200 TapeStation Controller' Software Controller Software Interface Assign a location to each samples you will be analyzing in the digital layout of the wells Select the wells [...]

DNA Fragment Analysis via 4200 Agilent TapeStation2019-07-08T18:59:38+00:00

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 transformation into yeast of a PCR amplicon containing the desired selectable marker or tag flanked [...]

gene deletion & gene fusion protocol for yeast2019-02-06T19:37:38+00:00

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 of a selectable marker flanked by DNA sequences homologous to the the last 40-60 bp of [...]

C-terminal tag protocol: Adding a C-terminal tag to a gene2019-02-06T19:37:38+00:00

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 of a selectable marker flanked by DNA sequences of 40-60 bp homologous to the 5' [...]

N-terminal tag protocol: Adding an N-terminal tag to a gene2019-02-06T19:37:38+00:00

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 concentrate it just by drying it down in the  Vacuum Centrifuge . However, if the DNA [...]

Phenol-chloroform extraction & EtOH precipitation protocol2019-02-06T19:37:38+00:00

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 in which the gene is replace with the kanMX selectable marker (G418 resistance). It is [...]

Primers for gene deletion in yeast (design protocol)2019-02-06T19:37:38+00:00

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. coli. For genomic DNA to be used for PCR amplification - such as testing for [...]

DNA extraction protocol for yeast genomes or plasmids2019-02-06T19:37:38+00:00

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 proteins are expressed at high concentrations. The principles of protein gel electrophoresis are similar to [...]

SDS-PAGE protocol: visualizing proteins on acrylamide gels2019-02-06T19:37:40+00:00