221 Transformation of E. coli with pGAL™ (blue colony) - EDVOTEK

24EDVO-Kit # 221Transformation of E. coli with pGAL (blue colony)Pre-Lab PreparationsPreparation of Competent CellsDay before the LabInstructor’s Guide1. Use a 10 ml sterile pipet to add 6 ml sterile cell reconstitution medium(Component F) to the vial of LyphoCells.2. Replace the rubber stopper and cap. Mix by inverting until thefreeze dried plug is dissolved.3. Shake the cell suspension vigorously and incubate the vial at 34-37°Cfor 16 - 24 hours (overnight) in an incubation oven. For optimalresults, incubate LyphoCells for 19 hours.Day of the Lab4. Completely thaw the competency induction solvent (G) and place onice. (If there is a white precipitate in the bottle, warm it in a 37°Cwaterbath to dissolve the precipitate.)5. Mix and resuspend the vial of incubated cells by inverting and gentlyshaking. Place the vial on ice for 10 minutes.6. Use a 10 ml sterile pipet to add 3 ml of ice cold competency inductionsolvent (G) to the vial of cells.The competency induction solvent is very viscous. Make sure that aportion of the solvent is not left on the walls of the pipet.7. Mix the cells and induction solvent thoroughly by inverting the vialseveral times. The solution should have no dense layers, "streams"or globules (i.e. it should be a uniform suspension).8. Keep the cells on ice for a minimum of 30 minutes.Cells can be kept on ice for up to 3 hours.Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratoryuse only. This document, or any part, may not be reproduced or distributed for any other purpose withoutthe written consent of EDVOTEK, Inc. Copyright © 1989, 90, 93, 94, 97, 98, 2000, 2005, EDVOTEK, Inc., all rightsreservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com