Protocols

/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

By |2019-02-06T19:37:38+00:00August 18th, 2017|

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 [...]

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

By |2019-02-06T19:37:38+00:00August 17th, 2017|

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 [...]

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

By |2019-02-06T19:37:38+00:00August 17th, 2017|

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' [...]

Phenol-chloroform extraction & EtOH precipitation protocol

By |2019-02-06T19:37:38+00:00July 28th, 2017|

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 [...]

Primers for gene deletion in yeast (design protocol)

By |2019-02-06T19:37:38+00:00July 28th, 2017|

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 [...]

DNA extraction protocol for yeast genomes or plasmids

By |2019-02-06T19:37:38+00:00July 28th, 2017|

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 [...]

SDS-PAGE protocol: visualizing proteins on acrylamide gels

By |2019-02-06T19:37:40+00:00July 26th, 2017|

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 [...]

agarose gel electrophoresis protocol: visualizing DNA

By |2019-02-06T19:37:41+00:00July 26th, 2017|

Peccoud lab Protocol: Visualizing DNA following size exclusion on an agarose gel Introduction In agarose gel electrophoresis, a sample of a biological molecule, in this case, DNA, is loaded into a well formed in an agarose gel matrix. To prevent the DNA from diffusing out of the wells of the gel, a small amount of [...]