Experiment Procedures - Frederiksen

Experiment ProceduresEDVO-Kit # 275AIDS Kit II: Simulation of HIV-1 Detection by Western Blot9SDS Protein-Agarose Gel ElectrophoresisPREPARATION OF ELECTROPHORESIS BUFFER AND AGAROSEGEL1. Dilute 1 part 10X Gel Buffer (Tris-Glycine) in 9 parts distilled water. Tomake 2.5% agarose in 1X Gel Buffer, determine volume of agaroserequired for your gel tray. Refer to the chart below to determinevolume required.2. Add the required amount of protein agarose powder to the requiredvolume of buffer. Swirl to disperse clumps.3. With a marking pen, indicate the level of the solution volume on theoutside of the flask.4. Heat the mixture to dissolve the agarose powder. The final solutionshould be clear (like water) without any undissolved particles present.a. Microwave method:• Cover flask loosely with plastic wrap to minimize evaporation. Donot cover tightly.• Heat the mixture on High for 1 minute.• Swirl the mixture and heat on High in bursts of 25 seconds until allthe agarose is completely dissolved.b. Hot Plate method:• Cover the flask with foil to minimize evaporation.• Heat the mixture to boiling with occasional stirring. Boil until theagarose is completely dissolved.5. Cool the agarose to 55°C with swirling to promote evendissipation of heat. If detectable evaporation has occurred,add hot distilled water to bring the volume of thesolution up to the original volume as marked on the flask.55˚CTable A: 2.5% SDS Protein Agarose GelSize of EDVOTEKCasting TrayAmt of Volume 1X Gel TotalAgarose + Buffer = Volume7 x 7 cm7 x 14 cm0.5 gm 20 ml 20 ml1.0 gm 40 ml 40 mlDuplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This document, or any part, may not be reproduced or distributed for any other purposewithout the written consent of EDVOTEK, Inc. Copyright © 2005 EDVOTEK, Inc., all rights reserved.EVT 004185AMThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com