GT CHEM 4581 - LECTURE NOTES (3 pages)

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LECTURE NOTES



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LECTURE NOTES

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Pages:
3
School:
Georgia Institute of Technology
Course:
Chem 4581 - Biochemistry Lab I

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Performing SDS PAGE CHEM 4581 Biochemistry Laboratory I Version February 2008 GOALS One of the goals of this week s lab is for students to learn how to pour run stain and destain an SDS PAGE gel For the purpose of teaching students will be given one or more protein samples in which to run on a gel TA s will be carefully watching student performance in order to correctly instruct students on the subtleties of performing this experiment Students will hopefully develop their skills in performing SDS PAGE for a direct application in next week s lab MATERIALS AND REAGENTS Mini Protean III Gel Electrophoresis Apparatus 1 5 M Tris HCl buffer pH 8 8 0 5 M Tris HCl buffer pH 6 8 10 Ammonium Persulfate 10 Sodium Dodecyl Sulfate 40 w v Acrylamide Bisacrylamide 35 1 molar ratio N N N N Tetramethylethylenediamine TEMED Laemmli Sample Buffer 10X Electrophoresis Running Buffer Coomassie Stain Destain Bio Rad Students will prepare this buffer for themselves Students will prepare this buffer for themselves Make fresh for each section Must add 2 mercaptoethanol to this buffer daily Must dilute to 1X before use Recycle stain solution EXPERIMENTAL PROCEDURES 1 Prepare each of the Tris HCl buffer solutions These solutions will be stored and used in a subsequent laboratory session 10 20 mL of 0 5M Tris HCl pH 6 8 20 30 mL of 1 5M Tris HCl pH 8 8 2 Clean and completely dry the glass plates combs and any other pertinent materials 3 Place a short plate on top of a spacer plate Insert both plates into the green casting frame on a flat surface Be sure that the legs of the casting frame are down Clamp the casting frame and check that the plates are level on the bottom 4 Put the casting frame into the casting stand 5 Combine all reagents except the APS and TEMED in the order listed in the table below for a 10 resolving gel Be sure to carefully add the acrylamide mixture to your solution in the fume hood Cover your 15 mL conical tube while transporting it back to your workbench 6 When ready to



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