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This wiki contains protocols and other information mostly of interest to the mec group at CBMA and people working in the LGM lab.
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Basics
- Starting to work in the MEC group
- Maintaining lab notes
- How to mark your material
- Autoclaving
- Yeast and bacterial freezer stocks
- LGM
- Congresses
- Dept phone directory
- Teaching
Commonly used protocols
- Yeast transformation
- E. coli transformation
- PCR
- Microwave Yeast Colony PCR ◀️ This is the fastest protocol
- NaOH Yeast Colony PCR ◀️ This more work, but robust
- Yeast genomic DNA extraction with LiOAc SDS ◀️ This more work, but robust
- Agarose gel
- Plasmid miniprep with home made buffers
- Plasmid rescue from yeast to E. coli
- Making LB medium.
- Making YPD medium.
- Making defined SC or SD medium .
Cultivating the yeast Saccharomyces cerevisiae for any purpose
The Yeast Pathway Kit
- The Yeast Pathway Kit
- pYPKa cloning protocol
- Transcription Unit cloning protocol
- standard_primers
- primer design
- in silico assembly of pYPKa_A_ATF1
- in silico assembly of pTA1_TDH3_ATF1_PGI1
- Plasmids, Promoters & Terminators sequence files.
Gel electrophoresis
Plasmid DNA from E. coli
- Boiling miniprep
- CAVAN low cost plasmid miniprep protocol without kit (super snik)
- Merlin
- Plasmid midiprep protocol
- Plasmid rescue from yeast to E. coli with plasmid miniprep kit
DNA
PCR
DNA extraction & colony PCR
Colony PCR from yeast is a notoriously fickle protocol which can be seen by the many protocol variations available online. It seems many users have to tweak their protocols for them to work. The protocols below are listed in order of difficulty. Test the protocol with a positive and negative control if you can.
- Microwave Yeast Colony PCR
- Yeast genomic DNA extraction with LiOAc SDS
- Yeast DNA isolation with Lyticase or Zymolyase
- Rapid isolation of yeast total DNA
- Extraction of chromosomal DNA with PCA (phenol:chloroform:isoamylalcohol)
Transformation
E. coli
S. cerevisiae
Yeast cell (protein) extract from S. cerevisiae
RNA
Miscellaneous Methods
- bradford
- sequencing
- Taq DNA polymerase purification from E. coli
- Yeast cell fixation with formaldehyde
E. coli strains
E. coli media
- LB
- LBpowder
- M9 (Dfined medium)
- SOB (Super Optimal Broth)
- SOC (SOB with glucose)
- Super Broth
- Terrific Broth
- LB24