Protocols

These protocols are updated relatively often (annually or so), and as we publish papers containing new methods the protocols should appear on this page.

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).

Protocols linked to recent papers

High mlecular weight DNA protocols including TrAEL-seq and END-seq: NEW July 2020

Embedding cells in agarose - NEW July 2020
Pulsed Field Gel Electrophoresis - NEW July 2020
Oligonucleotide 5' adenylation for linkers - NEW July 2020
Modified END-seq protocol - NEW July 2020
TrAEL-seq - NEW July 2020

Circular DNA sequencing protocols: NEW April 2020 for Hull et al. PLoS Biology 2019,e3000471

CircleSEQ (aka REC-seq) - NEW April 2020, please cite Hull et al. PLoS Biology 2019,e3000471
CircleSEQ SmaI (aka REC-seq) - NEW April 2020, please cite Hull et al. PLoS Biology 2019,e3000471
Low cell yeast DNA isolation - NEW October 2018, please cite Hull et al. PLoS Biology 2019,e3000471     

Yeast replicative ageing protocols: NEW October 2018 for Cruz et al. eLIFE 2018, e34081

Grow, label and harvest cells using the MEP system - updated July 2020, 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 - updated July 2020, please cite Cruz et al. eLIFE 2018, e34081

         

Our standard lab protocols

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:

Cloning - updated May 2018
PCR - updated May 2020
RT-PCR
PicoGreen quantification - updated July 2020
qPCR - NEW May 2018
 
Gel running, 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 - see Methods Mol Biol. 2020;2063:83-103
Denaturing PAGE- updated July 2020
In vitro transcription - updated May 2018
Ladder labelling
Probing blots - updated July 2020
Western blotting - updated May 2018
siRNA blotting

Library preparation protocols:

mRNAseq library preparation - NEW May 2018
KAPA quantification - updated July 2020
AMPure XP bead purification - NEW July 2020

Yeast work:
 
Yeast culture - updated May 2018
Yeast genetic engineering
Yeast Transformation - NEW May 2018
Harvesting yeast - updated May 2018
Synchronised meiosis - updated July 2020
BY4741 sporulation - updated July 2020
Yeast Colony PCR - updated May 2018
Yeast Spotting assay - NEW July 2020
Yeast DNA isolation - updated July 2020
Yeast PFGE - this is a legacy protocol replaced by the general PFGE protocol above and the agarose embedding protocol below
Yeast propidium iodide staining for FACS
ChIP for yeast
Yeast immunofluorescence
Yeast protein isolation - updated May 2018
TAP purification

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 July 2020
Yeast mirVANA total RNA prep - NEW May 2018
 

 

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.