Keogh Lab Protocols 


All lab protocols (with extensive notes) downloadable as PDF files

Arrangment is alphabetical within each subsection

 Note that all of these protocols are used in the lab

 

Updated 7.8.10


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  Lab Protocols (Miscellaneous)

  • Activated Na-Orthovanadate: Activation depolymerizes the vanadate, converting it to a more potent inhibitor of protein tyrosine phosphatases.
  • Chromatin: A collection of multiple protocols and a work in progress. #I. Expression and purification of recombinant S.cerevisiae H2A, H2A.Z and H2B.
  • Cloning PCR products: There are few things more boring than screening through huge numbers of empty plasmids to find a desired clone. This protocol suggests a number of controls that will immediately suggest where it’s all going wrong.
  • Immunoprecipitations: A standard protocol for immunoprecipitating from yeast WCEs. Includes information on antibodies used in the lab for this purpose and the binding characteristics of proteins A and G. IPs are used to concentrate factors for the identification of associated factors or downstream enzymatic reactions. To the latter end a sample protocol for the analysis of CDK (cyclin-cependent kinase) activity is also described.
  • Kinase assay: As used to assay the activity of an immunoprecipitated CDK (cyclin-dependent kinase) from yeast WCEs.
  • Media: Growth media and simple additives (carbon sources, etc) for culturing yeast and E.coli (for S.pombe use the specifc recipes in this supplemental protocol). Use in combination with Media Additives protocol. Yeast auxotrophies are well described in this review.
  • Media Additives: Antibiotics, cell-cycle arrest agents, counter-selection agents, genotoxins, HDAC inhibitors and transcription elongation inhibitors. Use in combination with Media protocol.
  • Recombinant TEV Protease: Expression and purification of HIS-tagged TEV (Tobacco Etch Virus) NIa protease with an S219N mutation that removes a self cleavage site.
  • Restriction enzyme digestion of PCR products: Used to determine whether a PCR amplicon is of the appropriate region. A full sequence, or at least a rudimentary restriction map, of the expected amplicon will be required. It’s preferable to employ a limited set of restriction enzymes (BamHI, EcoR1, EcoRV, HindIII or XbaI) since these are cheap efficient cutters.
  • RNA (misc): Protocols for preparing nuclease-free (DEPC-treated) reagents, isolating RNA from yeast (see also below), gel resolution (Agarose and PAGE), transfer to membranes (pressure and electrophoretic), Northern blotting, probe preparation (and size-exclusion chromatography for removing free nucleotide), and general info on working with this labile molecule.
  • RNA solubilization in formamide: Resuspending RNA in formamide has several benefits over storage in ddH2O or EtOH, including protecting it from nucleases and allowing higher concentrations.
  • RNAse A, DNAse-free: Preparation of; supplementary from the yeast Genomic DNA extraction and analysis protocols.
  • RT-PCR: cDNA synthesis with Superscript II Reverse Transcriptase. Used to examine parent mRNA splicing, termination, polyadenylation, etc.
  • Western Blotting: Immunodetection of Whole Cell Extracts (WCEs), immunoprecipitates, recombinant proteins, etc. Also contains information on antibodies in common use in the lab and a protocol for India Ink staining the membrane.

  Lab Protocols (S.cerevisiae)

  • beta-Galactosidase assays: The LacZ gene of E.coli encodes beta-Galactosidase, capable of the hydrolysis of a variety of beta-D-galactosides including chromogenic substrates (colorless compounds that yield a colored compound when hydrolyzed). Two assays are described: (i) the liquid assay (ONPG substrate) is sensitive, quantitative and generally used to accurately monitor the kinetics of gene expression. (ii) the filter assay (X-GAL substrate) is less sensitive and provides only a qualitative assessment of enzyme activity. However it is useful for simultaneously analyzing a large number of colonies.
  • Cell-cycle arrests: Protocols for arrest with alpha-Factor, Hydroxyurea, Nocodazole and cyclin-mutants. For synchronization studies alpha-Factor is preferable as the release is a lot cleaner. 
  • Cell Fractionation: In addition to fractionating S.cerevisiae for direct analyses, a variant of this protocol is used for purifying chromatin / nucleosomes. These are then directly analyzed or immobolized to beads for affinity purification of binding factors.
  • ChIP Primers: Primer sets for analysis of constitutive (ADH1, DBP2, PDR5, PMA1, PYK1, YEF3) and inducible (GAL1) genes, centromeres (CEN-III), Telomeres (TELV) and an induced DSB at MAT.
  • ChIP-chip: Goal is to randomly amplify a representative DNA sample and use this to probe a microarray. Not a “linear” method, but useful to compare relative enrichment between two samples. This protocol has been used successfully to amplify genomic representations of less than 10ng of DNA (used in: Kim et al  (2004) Nature 432:517).
  • Counterselection with α-AminoAdipic Acid (αAAA): Many yeast labs use 5-FOA as a counter-selection agent to URA+, but few use αAAA against LYS+, usually because they’ve heard it doesn’t work. Not so: it does, although those familiar with the FOA / URA system will not be overly impressed with αAAA / LYS. The requirement for replica plating and relatively high backgrounds are significant issues with the latter. However it is a useful backup option if URA is otherwise unavailable.
  • Fermenter runs: If you need a lot of yeast use a fermenter instead of conical flask culture: the cells can be grown to much higher densities and still remain in log phase. Under the conditions described the expected yields are 10-20g/L compared to the usual 3-5.
  • Galactose induction: For galactose induction of a protein of interest the gene is placed under  regulation of the GAL1-10 promoter and cells grown in the presence of 2% galactose as a primary carbon source. The protocol describes many of the time-course analyses possible with this inducible system.
  • Immunostaining: You’re going to look at these yeast under a microscope, so you need very few cells compared to usual. Includes steps for permeabilizing yeast cells, mounting onto poly-lysine slides and immunostaining for fluorescent detection.
  • Immobilized Template assays: For these assays the template is biotinylated, immobilized onto beads, and IVT-grade WCEs added. The biggest potential problem is high-background. Methods to reduce this include blocking with BSA, competition with random DNA or addition of the detergent Sarkosyl. However the best way to reduce background is to use as little WCE as possible: for this reason the greater the Tx activity, the better the extract.
  • In vitro Transcription (IVT): A collection of three protocols for: (A) making IVT-grade whole cell extracts (WCEs), (B) RNA Polymerase II (RNApII) assays, and (C) RNA Polymerase III (RNApIII) assays. All require significant optimization but are worth the effort. It is strongly advised to normalize RNApII activity (either between multiple preparations of the same strain or WT vs. mutants) by RNApIII activity rather than amount of protein. Related protocols: making recombinant Gal4-VP16 activator and TFIIS.
  • In vitro Transcription Elongation (Tailed Template): In this promoterless template the tail acts like a premelted transcription bubble. The reactions contain purified RNapII and any putative elongation factors you want to test the activity of. Pre-Initiation Complex (PIC) components are not required.
  • In vivo HA3 tagging: For the PCR amplification of an epitope-tag or deletion cassette from a plasmid. Use long PCR primers (≈75 bases) to amplify a cassette (≈25 bases of the primer) flanked by ≈50 bases of homology to a location of interest. This product is then transformed into S.cerevisiae and integrates with high efficiency by homologous recombination. Use in conjunction with the protocol for genomic DNA purification above.
  • Mating pheromone experiments: Includes protocols for switching the mating type of a strain and two ways to determine mating type: (i) the Halo assay (identifies the secreted mating pheromone), and (ii) the direct mating assay.
  • MMS mutagenesis: Used to further mutagenize a mutant strain and look for synthetic or bypass phenotypes. Important to balance the number of de novo mutations induced in each cell: too many and you’ll never be able to track down which one is interacting with your study mutation. A balance between the induced mutation rate and killing curve will help you guesstimate the appropriate exposure time.
  • Phenotypes: Describes a compendium of phenotypes that can be easily screened to identify pleiotrophic defects associated with a mutation. In many cases a particular phenotype (or set thereof) can suggest a function for the gene under study (Hampsey M (1997) Yeast 13:1099).
  • RNA extraction: with hot acidic phenol. Protocol is well suited for obtaining reproducible quantities of RNA largely devoid of contaminating DNA that partitions into the interface during the extraction step. See also the RNA protocols above.
  • Smash and Grab: A harsh treatment of yeast that yields plasmid DNA and sheared genomic DNA. This protocol is used to release plasmids from yeast, which are then transformed into bacteria for amplification. Usually done to rescue library plasmids and determine if the phenotype under study is plasmid linked.
  • Sporulation-dissection: >90% of the time we work with diploids, it is after mating two haploid strains and sporulation is intended. To track marker segregation two methods are possible; (i) tetrad dissection, or (ii) random sporulation.
  • Spotting: Used to analyze growth phenotypes, spotting takes longer than streaking, but is preferable since it gives much better pictures. 
  • TAP-purification, one-step: Gives great yields as used to purify kinases, phosphatases and HATs for in vitro analyses. If you want super-pure samples for MS-studies use the two step protocol (much lower yield, but higher purity).
  • TAP-purification, two-step: Gives very clean samples (as used when you want to identify complex components by Mass Spec). However the yield sucks (most protein remains on the calmodulin beads) so not recommended for making active enzyme complexes for functional studies. (For a description of the TAP tag, see the original paper; Puig et al (2001) Methods 24:218).
  • Transposon mutagenesis screen: Uses the Snyder Library (Burns et al (1994) Genes Dev 8:1087) to transposon mutagenize a large population for synthetic or bypass screens. Genomic coverage: ≈10,000 colonies, ≈90%; ≈30,000 colonies, ≈95%.
  • Whole Cell Extracts (II); suitable for IPs: NOT my preferred method for quick screening in vivo tagged proteins or looking at chromatin modifications. For those I use the TCA extraction method (below); quicker, easier and much more efficient at extracting chromatin. The IP protocol is described above.
  • Whole Cell Extracts (III); TCA method: Extremely efficient and probably more representative of the protein situation in the cell since it also extracts chromatin (based on the ability to see modified yeast histones). Also the TCA kills enzyme activity so you preserve labile modifications like phosphorylation or acetylation without the need for inhibitors. NB. You can’t IP from these extracts – they’re only for westerns. 
  • Whole Cell Extracts (IV); IVT-grade: in vitro transcription (IVT) grade extracts (also suitable for immobilized template studies). Adds an ammonium sulfate-cut after extraction to concentrate the proteins. Takes an entire day from the breaking step, and a long day at that. Save time by preparing all buffers the day before and store in the coldroom.

  Lab Protocols (Sz. pombe)

  • S.pombe requires longer homology than S.cerevisiae (>200bp vs. ~50bp) for efficient homologous recombination. The two-Step PCR protocol for deleting or C-terminal tagging of S.pombe genes. Includes a map of the primers, PCR conditions, sample primers for two genes (Swc6 and Ash2), and their location on the pFA6a.KanMX sequence. 
  • Simple protein extraction: As used for rapid screening / confirmation of TAP-tag integrants. Throughput is high as all manipulations are performed in the same tube. This protocol is not suitable if you want to look at chromatin components (particularly things like modified histones) - for that use the TCA extraction protocol.
  • More planned [1.21.10]

  Lab Protocols (E.coli)

  • Minipreps: Old school: I dislike this protocol – it takes longer and the DNA quality isn’t as good as Qiagen mini-prep (or suchlike) columns. It is NOT recommended when preparing a vector backbone for cloning: many restriction enzymes cut with <100% efficiency giving high backgrounds.
  • Recombinant Proteins #1: Expression and purification of HIS-tagged recombinants. The protocol attempts to recover soluble and insoluble fractions (some proteins will exclusively partition into one or the other phase). NB: Recombinant protein expression is generally done in specialized strains, distinct from those used for cloning - see E.coli strains list below).
  • Recombinant Proteins #2: Expression and purification of GST-tagged recombinants. Glutathione agarose is relatively cheap and it’s an easy matter to elute the recombinant protein by competition with free glutathione. For background info on the GST-system see the Amersham GST-handbook. NB: see also E.coli strains list below.
  • Recombinant TEV Protease: Specific protocol for the expression and purification of HIS-tagged TEV (Tobacco Etch Virus) NIa protease with an S219N mutation that removes a self cleavage site.
  • Strains: 2 x E.coli strain genotype tables: first from NEB, second from Novagen. The latter gives detailed information on BL21 derivative strains used for recombinant protein expression.

  External Resources

  • Haber lab: Protocols for the analysis of DNA double-strand break (DSB) repair in Saccharomyces cerevisiae
  • Forsburg Lab: Everything you need to know about working with Schizosaccharomyces pombe
  • OpenWetWare: Continuously updated protocols on everything from E.coli  to Microfluidics
  • Promega: Protocols and guides. Great source of info on cloning and restriction enzymes and lots more