02 - Code Breakers: Using CRISPR to Rewrite Genetics
Experiment Procedure: Transformation
150 µL
1
REMOVE 250 µL
2
MIX
5000 rpm
ROTATE & SPREAD
SPREAD the cells
VISUALIZE and RECORD
48 hours @ 37°C
COVER
9. While cells are recovering, COLLECT and LABEL the bottom of two agar plates as indicated below. NOTE: Keep writing small and along the edge so you can easily see your colonies at the end. • 1 or "PG" for pCas9-PurpleGuide • 2 or "CG" for pCas9-ControlGuide 10. Following recovery, SPIN the tubes at 5000 rpm for 1 minute. 11. PLATE your two transformed cell lines by performing the following five steps one tube at a time. Use a new sterile pipet tip and loop for each tube/plate. (a) REMOVE 250 μL of the media. Work carefully during this step to making sure the cell pellet stays in place. (b) Gently RESUSPEND the pellet in the remaining media by pipetting up and down several times until no clumps are visible. (c) COLLECT 150 μL of the resuspension and PIPET into the center of the ap- propriate agar plate. (d) Use a loop to SPREAD cells evenly and thoroughly over the entire surface. (e) TURN the plate 90° and thoroughly SPREAD again. 12. COVER the plates with lids and INCUBATE at room temperature for 5 minutes to allow the liquid to be fully absorbed. 13. STACK the plates on top of one another and TAPE them together. INVERT plates (agar side on top) and PLACE in a 37°C bacterial incubation oven. INCUBATE for 48 hours. If you do not have an incubator, colonies will form at room tempera- ture in approximately 48-72 hours, but an incubator is HIGHLY recommended. 14. VISUALIZE the transformation and control plates and RECORD the number of colonies on the plate, the color(s) of the colonies (such as dark purple, light purple, white), and the number of colonies of each color. If the colors are faint, the plates can be left in the refrigerator (4°C) for 24-48 hours to for further color development.
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