Protocols
Many of these protocols have been updated and new ones were added in May 2018 and updated again in September 2018. Over the next year there will be further additions particularly for replicative ageing cell work and library preparation.
We have published detailed versions of our protocols for Northern blotting (Methods Mol Biol. 2020;2063:83-103) and PFGE analysis (Methods Mol Biol. 2013;1054:195-207).
Basic protocols for new lab workers:
Making solutions
Handling enzymes
Agarose gels for DNA - updated May 2018
Basic rules for RNA work
Standard molecular biology protocols:
https://link.springer.com/protocol/10.1007%2F978-1-4939-9822-7_5
Cloning - updated May 2018
PCR - updated September 2018
RT-PCR
PicoGreen quantification
qPCR - NEW May 2018
Blotting and probing protocols:
Genomic Southern Blot - please cite Hull et al PLoS Biol. 2017 Jun 27;15(6):e2001333 if you use this protocol
Glyoxal gel
Denaturing PAGE
In vitro transcription - updated May 2018
Ladder labelling
Probing blots - updated May 2018
Western blotting - updated May 2018
siRNA blotting
Library preparation protocols:
mRNAseq library preparation - NEW May 2018
KAPA quantification - NEW May 2018 - updated September 2018
Yeast work:
Yeast culture - updated May 2018
Yeast genetic engineering
Yeast Transformation - NEW May 2018
Harvesting yeast - updated May 2018
Synchronised meiosis
BY4741 sporulation - updated September 2018
Yeast Colony PCR - updated May 2018
Yeast DNA isolation
Yeast PFGE - updated May 2018
Which RNA prep do I use? Probably hot phenol, but GTC can be better for ribosome analysis. The GTC miniprep is the fastest and most consistent if you need to prep many samples and aren't too fussy about perfect quantification. With few samples, consider mirVANA kits – these produce beautiful RNA but are expensive
Yeast hot phenol RNA prep
Yeast hot phenol RNA miniprep
Yeast GTC RNA prep (Tollervey lab standard prep)
Yeast GTC RNA miniprep - updated May 2018
Yeast mirVANA total RNA prep - NEW May 2018
Yeast propidium iodide staining for FACS
ChIP for yeast
Yeast immunofluorescence
Yeast protein isolation - updated May 2018
TAP purification
Yeast replicative ageing protocols: NEW October 2018
Grow, label and harvest cells using the MEP system - NEW October 2018, please cite Cruz et al. eLIFE 2018, e34081 in addition to the original Gottschling lab paper if you use these protocols
Low cell yeast RNA isolation - NEW October 2018, please cite Cruz et al. eLIFE 2018, e34081
Low cell yeast DNA isolation - NEW October 2018, citation forthcoming
Circular DNA sequencing protocols: NEW April 2020
CircleSEQ (aka REC-seq) - NEW April 2020, please cite Hull et al. PLoS Biology 2019,e3000471
These are our in-house protocols - they are provided as is and we accept no responsibility for their use or mis-use. Please observe your organisation's health and safety policy while performing these procedures.