Transformation of E. coli with Green and Blue ... - Frederiksen

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12EDVO-Kit # 222Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsLaboratory SafetyImportant READ ME!The ExperimentTransformation experiments contain antibiotics which are used for theselection of transformed bacteria. Students who have allergies to antibioticssuch as penicilllin, ampicillin, kanamycin or tetracycine shouldnot participate in this experiment.1. Gloves and goggles should be worn routinely as good laboratorypractice.2. Exercise extreme caution when working with equipment which isused in conjunction with the heating and/or melting of reagents.3. Do not mouth pipet reagents - use pipet pumps or bulbs.4. The E. coli bacteria used in this experiment is not considered pathogenic.Although it is rarely associated with any illness in healthyindividuals, it is good practice to follow simple safety guidelines inhandling and disposal of materials contaminated with bacteria.5. Properly dispose materials after completing the experiment:A. Wipe down the lab bench with a 10% bleach solution or a laboratorydisinfectant.B. All materials, including petri plates, pipets, transfer pipets,loops and tubes, that come in contact with bacteria should bedisinfected before disposal in the garbage. Disinfect materialsas soon as possible after use in one of the following ways:• Autoclave at 121° C for 20 minutes.Tape several petri plates together and close tube caps beforedisposal. Collect all contaminated materials in an autoclavable,disposable bag. Seal the bag and place it in a metal tray to preventany possibility of liquid medium or agar from spilling intothe sterilizer chamber.• Soak in 10% bleach solution.Immerse petri plates, open tubes and other contaminated materialsinto a tub containing a 10% bleach solution. Soak the materialsovernight and then discard. Wear gloves and goggleswhen working with bleach.6. Wear gloves, and at the end of the experiment, wash hands thoroughlywith soap and water.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 without thewritten consent of EDVOTEK, Inc. Copyright © 1999, 2003, EDVOTEK, Inc., all rights reservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com
Transformation of E. coli with Green andBlue Fluorescent Protein PlasmidsEDVO-Kit # 222 13Transformation of E.coliSetting up the Transformation and ControlExperimentReminder“+”“-”- DNAContains oneor both plasmidDNAs+ DNADoes not containplasmid DNA1. Label one microcentrifuge tube "+ DNA". This will be thetransformation tube with one or both plasmid DNAs.2. Label a second microcentrifuge tube "– DNA" . This will bethe experimental control tube without plasmid DNA.3. Using a sterile 1 ml pipet, add 250 µl (0.25 ml) of ice cold CaCl 2solutionto each tube.4. As done in research laboratories, pick colonies from the source plateof E. coli cells. Use the following method for each of the test tubeslabeled "+ DNA" and "– DNA":• Use a sterile toothpick to transfer several colonies (2-4 colonies,approximately 2-4 mm in size) from the source plate to the testtube.• Between your fingers, twist the toothpick vigorously and upand down in the cold CaCl 2solution to dislodge the cells.The ExperimentSource PlateE. coli Cells5. In both tubes, suspend the cells completely by tapping or vortexing.6. To the tube labeled "+ DNA", add one of the following options:• 10 µl of pFluoroGreen(from tube labeled "pFG")• 10 µl of pFluoroBlue(from tube labeled "pFB")- DNATransfer 2-4 largecolonies to eachtube of ice cold+CaCl 210 µl+ DNApFluoroGreen and/orpFluoroBlue• 5 µl of each(a total volume of 10 µl)250 µlCaCl2Avoid scraping up agar when transferring the cellsfrom the source plate to the tubes with calciumchloride solution. It is important that the cells areresuspended in the calcium chloride solution and isnot left on the toothpick or on the wall of the tube.Proper experimental techniqueis critical - the plasmid DNA(pFluoroGreen or pFluoro-Blue) must be added directlyinto the cell suspension.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 without thewritten consent of EDVOTEK, Inc. Copyright © 1999, 2003, EDVOTEK, Inc., all rights reservedEVT 005077KThe Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com
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Transformation of E. coli with Green and Blue ... - Frederiksen

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