Hi! This post is going to be about...my crispr lab.
So first there are 4 stages ; 1 week before, 3 days before, 1 day before, and the day of the lab.The point of the experiment is to break the DNA then substitute it to make the fungus blue, instead of white.This is a picture of what it should look like when it is done:
A. MAKE PLATES
These are the steps for day one:
1. Thaw the Chix mix (place the tube in an incubator or heat block
set at 37°C.)
2. Add 190 ml distilled water to a heat-proof bottle
3.Add 7.5 g LB agar powder to the distilled water
4. Loosely cap the bottle.
5. Microwave in 30-second increments just until the solution boils. Continuously monitor to
ensure the solution doesn’t boil over.6. Using a heat proof mitt, carefully swirl the solution.
7. Repeat steps 5 and 6 at least two more times. Continue until the LB agar powder is fully
dissolved and the solution is transparent.
8. Let the LB agar solution cool until you can touch the bottle with your bare hands, but not so long that the agar begins to set. This should take approximately 3-5 minutes.
9. Mix the ChIX, then add 475 μl to the LB agar solution and swirl to mix.
Note: ChIX may refreeze at room temperature. Make sure solution is fully thawed before using.
10. Pour LB agar into sterile Petri dishes.
11. Immediately put the lids on the Petri dishes and allow LB agar to solidify.
(For a class with eight lab groups, you will need 16 plates total. Repeat steps 2-11 to pour
another set of eight plates.)
12. Once agar is solid, stack plates upside down with the agar on top.
13. Place stacked plates in a plastic bag and store in the refrigerator until use.
Because X-gal present in the ChIX is light sensitive, protect plates from light if possible.
B. REHYDRATE
BACTERIA
1. Uncap the vial of lyophilized bacteria.
2. Add 900 μl of LB + ampicillin liquid media to the vial.
3. Reinsert the rubber stopper to cap the vial and invert
several times to mix.
4. Aliquot 50 μl of rehydrated bacteria into 16 sterile
1.7 ml microtubes two tubes per lab group.
5. Incubate at room temperature (18-25°C) for 48-60 hours.
Do not disturb or agitate samples during the incubation
period.
C. ADD CaCI2
TO BACTERIA
1. Add 25 μl of sterile CaCl2
to each tube of bacteria.
2. Place tubes in refrigerator and chill for 12-24 hours.
 (12 hours recommended).
D. PREP SUPPLIES
& EQUIPMENT
1. Prepare the equipment
• Set water bath or dry bath to 42°C for heat shock.
Note: If using a dry bath, I recommend adding water to
the holes.
• Set incubator to 37°C for recovery after transformation
and incubating LB agar plates.
• Thaw tubes containing the DNA samples by placing them
on a rack or water bath at room temperature.
• For each lab group, dispense the following reagents into
labeled sterile 1.7 ml microtubes:
° pCtrl DNA, 30 μl
° pKO DNA, 30 μl
° SOC recovery media, 120 μl
3. Prepare ice
• We recommend using shaved or crushed ice rather than
large ice cubes.
• If starting with large ice cubes, smash ice until you have
pea-sized fragments.
4. Pre-warm ChIX LB agar plates
• Up to one hour before class starts, place plates in the
37°C incubator.
Here is the link to the knockout pdf;