KIELKOPF LAB PROTOCOLS


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General

Perkin Elmer Lambda25 UV/ VIS Spectrophotometer

Turning off UV/ VIS Spectrophotometer Lamps

Washing Glassware

Using Autoclave

Ordering from Roche

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Protein Purification and Biochemistry Protocols

Care for Akta Purifiers

E. coli Large Scale Induction of Protein Expression

Lysis of E. coli by Sonication

Protein Purification Buffers

Purify a Protein with Acidic pI

Purify a Protein with Basic pI

Purify a Protein with Thrombin Cleavage Site

Selenomethionine Protein Purification

A280 Protein Concentration Determination

Dialysis Tubing

Laemmli Method for SDS-PAGE Gels and Buffers

Tricine Gels

Western Blot

Prepare Sigma glutathione agarose

Expression Test

GST Pull-Down

GST Competition Pull-Down

Precision Protease Purification and Use

TEV Protease Purification and Use


U2AF65 DLink Purification

SF1 Purification

SF1 Mystery Domain Purification

Full length histag-U2AF65 Purification

Full length GST-ATPase Purification

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Nucleic Acid Protocols

DNA Desalting 2g SepPak

DNA Desalting 200mg SepPak

Nitrocellulose Filter Binding

Electrophoretic Mobility Shift Assays

Adjusting PhosphoImager Image Quality

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Thermodynamic Protocols

BIAcore T100 for kinetic or affinity measurements

Isothermal Titration Calorimetry (ITC)

Circular Dichroism Protocol

Fluoromax-3 Polarizer Alignment

Fluorescence Anisotropy RNA Binding Assay

Equations for Fitting Fluorescence Titration Assay

Electrophoretic Mobility Shift Assay

Amino Acid Analysis to Confirm Concentration for ITC

Effect of Interdomain Linker on Kd

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Structural Protocols

Dynamic Light Scattering (DLS)

Small Angle X-ray Data Collection at ALS (with Greg Hura's help)

Scaling Pr functions calculated from SAXS data

Running MONSA on SAXS data

Bruker CCD login information

Crystal Photos

Crystal Flash-Cooling Tips

Remote Data Collection at SSRL

Prepare SeMet Protein

Sazaki Table of f' and f"

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Subcloning Protocols

Making LB for Plates and Liquid Media

Antibiotics Guide

Single site mutagenesis

PCR Primer Design for Subcloning

PCR

Restriction Enzyme Digest of DNA Insert and Vector

Gel Purifying Your PCR Product

Photographing DNA gels

Ligation

Bacterial Transformation

Colony PCR

Overnight Cultures of successful transformants

DNA prep for sequencing

DNA sequencing at UR

Freezer stocks of transformed bacteria

Making Competent Cells

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