02 - Code Breakers: Using CRISPR to Rewrite Genetics
Experiment Procedure: Transformation
150 µL
1
2
REMOVE all but 150 µL
3
4
MIX
5000 rpm
VISUALIZE and RECORD
ROTATE & SPREAD
SPREAD the cells
COVER
37 °C OVERNIGHT
9. While cells are recovering, COLLECT and LABEL the bottom of four agar plates as indi- cated below. NOTE: Keep writing small and along the edge so you can easily see your colonies at the end.
• Purple pCas-Purple or 1 (Amp/IPTG/Chloramphenicol plate) • Purple pCas-blue or 2 (Amp/IPTG/Chloramphenicol plate) • Blue pCas-Purple or 3 (Xgal/IPTG/Chloramphenicol plate) • Blue pCas-blue or 4 (Xgal/IPTG/Chloramphenicol plate) 10. Following recovery, SPIN the tubes at 5000 rpm for 1 minute. 11. PLATE your four transformed cell lines by performing the fol- lowing five steps one tube at a time. Use a new sterile pipet tip and loop for each of tube/plate. (a) REMOVE all but 150 μL of the media*. Work carefully during this step to making sure the cell pellet stays in place.
*STEP 11 NOTE: Estimate 150 μL using the 0.1 mL volume marker on the tube. If necessary, overestimate and leave slightly more than 150 μL during steps 11a and 11b rather than underestimate.
(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 appropriate agar plate. (d) Use a loop to SPREAD the 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 overnight (24 hours). If you do not have an incubator, colonies will form at room temperature in ap- proximately 48 hours. 14. VISUALIZE the transformation and control plates and RECORD the number of colonies on the plate and the color(s) of the colonies. 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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