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: added July 2020, please cite Kara et al. PLoS Biology 2021,e3000886

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

Felix's GitHub page - software for TrAEL-seq data analysis

Here are some videos showing how to make and handle agarose plugs (thanks Anna and Alex for these!)

Making plugs - NEW August 2021
Cell lysis in plugs - NEW August 2021
Replacing solutions - NEW August 2021
General handling of plugs - NEW August 2021
Moving plugs to and from Falcon tubes - NEW September 2021
Replacing wash buffer - NEW September 2021

Transcriptome analysis of fixed, stained and sorted cells: NEW October 2020

Glyoxal fixation for RNA extraction from stained and sorted cells - NEW October 2020, please cite Channathodiyil and Houseley PLoS One 2021, e0240769

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

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

Yeast replicative ageing protocols: added 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 - added 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 - added May 2018 - a bit outdated now and an Ultra II version will be up soon
KAPA quantification - updated July 2020
AMPure XP bead purification - added July 2020

Yeast work:
 
Yeast culture - updated May 2018
Yeast genetic engineering
Yeast Transformation - added 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 - added 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 - added 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.