Biotechniques BIOL 410 Column Chromatography Chromatography Examines components
Biotechniques (BIOL 410) Column Chromatography
Chromatography • Examines components that give matter its “characteristics” • Relies on separation of components due to chemical/physical properties • Size • Polarity 2
Chromatography • Paper and Thin plate chromatography separate pigments • Column Chromatography – DNA separation – Protein separation • Column chromatography is often used to purify samples 3
Types of Column Chromatography • Exclusion chromatography – Prevents certain molecules from passing through & exiting the column – Cause different molecules to travel at different rates; leaving the column at different times • Affinity chromatography – Bind molecules, using polarity, to prevent them from passing through & exiting the column – Using elutants, overcome polar bonds to release molecules 4
High Performance Liquid Chromatography (HPLC) 5
HPLC • HPLC regularly use to separate protein components – Subunits 6
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Mini Column Chromatography Cap Reservoir Silica Filter Spout (for Elutant removal) 8
Spin Mini Column Chromatography • Use gravitation forces to help separate molecules – Along with Elution (polar) solutions 9
Vacuum Mini Column • Instead of using gravitation force • Pull sample through column with a vacuum – Desire produce need to be extracted separately • Same yield 10
Spin Columns • Spin columns are designed to fit into a capless microtube. • Tubes will then fit a microfuge or centrifuge with microtube rotor – We will use both 11
Column Components Mobile Phase • Solvent carrying desired solute • Weaker solvent; lower polarity Stationary Phase • Polar substance • Binds solute 12
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Elution • Washes with buffers of increasing polarity – Overcome charge-based bonds between solute and stationary phase • Washes with buffers remove unwanted material first – Then once unwanted material is gone, with release desired product, typically smaller molecules. 14
Purified DNA 15
Our Target • Bacteria transformed to carry Ampicillin resistance – Plasmid • We will isolate that plasmid from the cell 16
4 Step Process Column chromatography is a four-step process: 1. Lysis of the cells – To extract DNA 2. Binding – Adhere DNA to stationary phase 3. Washing – Remove unwated material 4. Elution – Collect desired product 17
Process 18
Today’s Lab • Collect Bacteria – Determine amount of bacteria using OD 600 – Make sure you have enough bacterial to get a good sample of DNA • Collect plasmid using mini column chromatography • We will save DNA and analyze next week! 19
Today’s Lab - Update • The bacteria carrying the plasmid did not grow • We will be collecting whole genome DNA from E. coli – Follow the process for determining bacterial density, and collect the optimal amount of bacteria (12 OD) – Follow Qiagen protocol for Lysis through column purification • Place samples on Ice (we will use next week) – Make sure to label with initials. 20
Abbreviations • P 1 – Pronase 1 – Lysis Buffer • P 2 – Pronase 2 – Lysis Buffer 2 • N 3 – Neutralization Buffer • PB – Binding Buffer • PE – Pre-Elution Buffer • EB – Elution Buffer 21
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