221 Transformation of E. coli with pGALâ¢ (blue colony) - EDVOTEK

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28EDVO-Kit # 221Transformation of E. coli with pGAL (blue colony)Experiment Results and AnalysisIdealized schematic of results.Instructor’s GuideX-GAL/Control 1Incubateovernight in 37Cincubation ovenAMP/X-GAL/Control 2Incubateovernight in 37Cincubation ovenAMP/X-GAL/pGALX-GAL/Control 1AMP/X-GAL/Control 2AMP/X-GAL/pGALWhite colonies(Lawn of bacteria)No coloniesBlue coloniesX-GALplated with controlcells (no DNA)Result: Plate coveredwith white colonies.May look like a smearedlayer or lawn of cells.Demonstrates:Colonies are whitebecause the cells do notutilize X-Gal. They donot contain pGAL DNAwhich contains a genethat will allow the cellto have a functional b-galactosidase.AMP/X-GALplated with controlcells (no DNA)Result: No growthDemonstrates:Host cells are sensitiveto ampicillin. WithoutpGAL DNA, they arenot ampicillin-resistant.They do not make b-lactamase.AMP/X-GALplated with cells+ DNA (pGAL)Result: Blue coloniesthat may have whitesatellite coloniesDemonstrates:1) A small portion of thecells are transformed;2) Cells are transformedwith pGAL and thereforecan utilize X-GALto give a blue color; 3)Cells acquire pGAL DNAand therefore ampicillinresistance.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
Transformation of E. coli with pGAL (blue colony) EDVO-Kit # 221 29Study Questions and Answers1. Did you observe any satellite colonies? Why are the satellite,feeder colonies white?Not all cells become transformed. Some of the untransformed cellscan grow around the colonies containing transformed cells. Thetransformed cells secrete b-lactamase which clears the surroundingmedium of ampicillin. Therefore, some of the untransformed cellsgrow. The satellites are white since they did not incorporate pGALDNA which contains the gene that will allow the cell to have a functionalb-galactosidase.2. Why did the competent cells which did not receive DNA (control)fail to grow on the plates containing ampicillin?Without pGAL DNA, they are not ampicillin-resistant because theydo not make b-lactamase.3. Why are there so many cells growing on the X-GAL plate? Whatcolor are they?Cells have not been challenged with antibiotic and therefore virtuallyall survive. They are white in color because they do not havepGAL DNA, and therefore, do not have a functional b-galactosidase.Instructor’s Guide4. What evidence do you have that transformation was successful?A successful transformation will show colonies on the plate labeledAMP/X-GAL/pGAL. An unsuccessful transformation will not showany colonies.5. What are some reasons why transformation may be unsuccessful?Unsuccessful transformations could be the result of many things,including: 1) not adding the plasmid to the host cells in the + pGALDNA tube, or 2) not adding a colony of bacteria to the + pGAL DNAtube, and 3) improper timing of the heat shock step.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
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221 Transformation of E. coli with pGALâ¢ (blue colony) - EDVOTEK