Methods from the Hahn Lab

Biochemistry Methods

Acetone precipitation of protein

BioRad Protein Assay

Brf1 purification from E. coli

Comassie Blue Protein gel stain

CNBr protein digest

Crosslinking of PICs and IP

Fluorescence polarization guide

E. coli total protein for SDS PAGE

3x Flag peptide elution

Gcn4 purification

Gel mobility shift assay (-Mg)

Immobilized Template Assay (Pol II)

Invitrogen NuPAGE and western transfer

Mobile Pilot Plant

Ni Sepharose (GE) manual

PEAS labeling of Gcn4

Pet_SUMO manual (Invitrogen)

Pol II yeast nuclear extract prep (small scale)

Pol II in vitro transcription and primer extension assay

Pol III in vitro transcription

Pol III yeast whole cell extract (small scale)

Protein Expression-PET plasmid handbook (Novagen; EMB Biosciences)

Silver Stain for SDS PAGE

Talon Metal affinity column purification

TCA precipitation of proteins

TFIIA Purification

TFIIIC purification from yeast

Yeast Nuclei isolation

Yeast protein prep for SDS PAGE and western (rapid)

Yeast small scale whole cell extract for IP

Western blot Probing and Stripping using dye conjugated antibodies (Odyssey)

Western Blot (Li-Cor Biosciences web resources)

Western blot probing by Chemiluminescence

Genetic Methods

Convert yeast A600 to cells/ml

EMS mutagenesis of yeast

Yeast Plates

Yeast Sporulation

Yeast spot test assay

Yeast transformation with DMSO/LioAc (high efficiency)

Yeast transformation (semi rapid)

Yeast Methods from the Gottschling lab

Molecular Biology Methods

Antibody Production using denatured proteins

Chromatin IP assay (Reeves/Tsukiyama)

Chromatin IP assay (Fishburn)

DNA concentration assay with fluorescent dye

DNA sequencing (dye terminator) [FHCRC form]

DNA sequencing (rapid Maxam and Gilbert Chemical degradation)

DNA Plasmid mini Prep

Electrocompetent E. coli preparation

Gels native Acrylamide, TBE (35 ml)

Gels (DNA sequencing, 100 ml)

Gels Acrylamide urea (35 ml gel)

in vitro mutagenesis

in vitro transcription and translation with PCR DNA

Oligonucleotide labeling with 32P

Oligonucleotide purification by PAGE and DEAE

PCR with Taq Polymerase (Promega)

PCR with expand Polymerase

PCR using yeast colonies

Primer extension of total Yeast RNA

Quantitative measurement of yeast mRNA using RT qPCR 

qPCR plate and master mix template (power sybr) 

qPCR 384 well Applied Biosystems SDS triplicate sample template

Quickchange mutagenesis with KOD extreme Pol

Quickchange site directed Mutagenesis (Pfu Pol)

S1 mapping with of yeast RNA with oligonucleotide probe

Single stranded dut- ung- DNA preparation

Yeast DNA preparation (rapid glass bead prep)

Yeast genomic DNA Preparation (high quality DNA)

Yeast RNA (acid phenol) SM induction and cDNA synthesis

Yeast quick plasmid DNA prep

Proprietary Methods (password required)

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