02 - Code Breakers: Using CRISPR to Rewrite Genetics
Experiment Procedure: Transformation
1
2
1
2
150 µL ice-cold CaCl 2
10 µL Plasmid (See table 2)
MIX
MIX
30-60 min @ 37°C
45 sec. @ 42°C
1
2
150 µL SOC to each tube.
Gently flick to mix.
NOTE: Keep tubes on ice as much as possible during this module. 1. COLLECT your two tubes of competent cells. LABEL them "1" and "2". ENSURE that the cells are kept on ice at all times. 2. ADD 150 µL ice cold CaCl 2 solution to each tube and MIX by gently pipetting up and down several times. 3. Using a new pipette tip for each tube, ADD 10 µL of the appropriate plasmids to each tube (see Table 2). MIX by gently pipetting up and down several times. 4. INCUBATE tubes on ice for 15 minutes. 5. Quickly PLACE the transformation tubes in a 42°C water bath for exactly 45 sec- onds. 6. Immediately RETURN the tubes to the ice bucket and INCUBATE for 2 minutes. 7. Using a new pipette tip for each tube, ADD 150 µL of SOC solution to each tube. MIX by gently flicking each tube. 8. PLACE tubes in a 37°C water bath and INCUBATE for 30-60 minutes to allow for recovery. continued
TABLE 2: Experiment Overview
Bacteria Strain
Plate
Plasmid
Experiment 1 2
E. coli Purple E. coli Purple
Amp/IPTG/Chloramphenicol Amp/IPTG/Chloramphenicol
pCas9-PurpleGuide “PG” pCas9-ControlGuide “CG”
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