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Unformatted text preview: BIOL 519 Week # 12- LacZ fusions- Transform and Identify Candidates BACKGROUND Last week we began the “LacZ fusions” experiments that we will continue this week. You used PCR to amplify the rhaBAD promoter region and then ligated the product into the plasmid vector pRS414. On Tuesday of this week you will transform those ligations into competent cells of strain DH5α (the same strain you used in week 5). The transformations will be plated onto LB plates containing X-gal, L-rhamnose and ampicillin. The ampicillin will select for the pRS414 plasmid, which encodes ampicillin resistance. The L-rhamnose will induce the rhaBAD promoter, so that any cells carrying the desired fusion will be in the induced state. Finally the X-gal will allow us to identify potential clones since they should be blue in the presence of L-rhamnose. Then on Thursday you will streak colonies that are blue on the X-gal L-rhamnose plates onto media with and without L-rhamnose. We know that the rhaBAD promoter is turned off in the absence of L-rhamnose, and only turned on in the presence of L-rhamnose. Since only very few other promoters have this same response, we can be quite sure we have the correct clones if we choose ones that respond appropriately to L-rhamnose. 1 BIOL 519 Week # 12- LacZ fusions- Transform and Identify Candidates TUESDAY
Experiment 9 - LacZ fusionsObjective: • Transform ligations Transformation 1. Thaw 6 tubes of competent cells provided by the TA. When thawing competent cells, you must make sure they do not get warm - as soon as liquid begins to appear, gently tap/mix the tubes. This will rapidly thaw the cells, and they should immediately be placed on ice. 2. Thaw your ligation reactions, but before removing any aliquots, spin them in the eppendorf centrifuge for 10 seconds to get all the liquid to the bottom of the tube, and then gently mix the reaction. 3. Add each of your 5 ligations (20 µl) to the 1st 5 tubes of competent cells (label carefully) and label the 6th tube “ND” for No DNA control 4. Incubate on ice for 30 minutes. 5. Heat shock the cells by placing the tubes in a 42oC water bath for 90 seconds and then chill them by immediately returning them to ice. 6. Add 800 µl of LB medium to each of the transformation tubes (including the no DNA control). 7. Incubate the tubes at 37oC for 30 to 60 minutes. (While this is incubating, label all of the plates that you will plate your transformations onto) 8. Spin down the cells in each of your tubes at maximum speed for 30 seconds in the microcentrifuge. 9. Pour off all but the last 100 to 200 µl of the supernatant 10. Gently resuspend each pellet in the remaining LB 11. Plate each transformation onto LB Ampicillin X-gal rhamnose. Label the plates carefully before plating the transformations. Double check that you are plating the correct transformation onto the correct plate. 12. Incubate plates at 37oC for 18-24 hours The TA’s will remove your plates on Wednesday Summary of expected transformation results: Ligation Expected # Colonies 1. Double cut vector very low 2. Double cut + ligase High 3. Double cut + Insert + ligase Hopefully, higher than #2 4. Double cut + Insert + ligase Hopefully, higher than #2 5. Uncut Very High (100’s to 1,000’s) 6. No DNA zero Phenotype of Colonies White White Some blue Some blue White zero 2 BIOL 519 Week # 12- LacZ fusions- Transform and Identify Candidates THURSDAY
Experiment 9 - LacZ fusionsObjective: • Choose candidates for rhaB-lacZ fusions and streak them +/- L-rhamnose. Streak out 8 candidates for your rhaB-lacZ fusion onto two plates each: (Streak 4 candidates per plate, therefore you will need two of each of the plates) • LB X-gal Ampicillin L-rhamnose • LB X-gal Ampicillin -You should pick blue colonies that came out of ligations 3 and/or 4 (the ligations that included insert DNA). The only plates with blue colonies should be plates from ligations 3 and 4. -Streak 4 candidates per plate -Pick one colony and draw the 1st line of your streak on each of the two plates with that one toothpick. Then go back and do the rest of the streaking with new toothpicks. -Make sure that your plates are well labeled so that you can identify which ones came from the same colonies. NOTE: TAs – make sure all groups have 2 candidates that are blue (+) L-rhamnose and white (-) L-rhamnose when you remove the plates! 3 ...
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- Spring '10