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

• 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

• EMS mutagenesis of yeast

• Yeast Plates

• Yeast Sporulation

• 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 extraction from Agarose (spin column)

• DNA sequencing (dye terminator)

• 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

• Quick Change site directed Mutagenesis

• 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 acid phenol RNA Preparation

• Yeast quick plasmid DNA prep