DIY Bacterial CRISPR 

make a regular LB Agar plate
streak it with DH5A and incubate at 37 for EC growth period


make 2 LB Strep/Kan/Arabinose Agar plates (all quantity in 150 ml water?) with microwave the same as any Agar plate. Keep it at 4C.


Pipette 100uL of Transformation mix (PEG 8000 and CaCl2) to a CT 
scrape a full innoculation loop off the DH5A? plate and mix in the transformation Mix CT. Put enough and mix until cloudy
store at 4C until needed. This becomes competent cell

Perform main reaction
new tip and pipette 10ul of Cas9 in your CT containing competent cells. Eject tip
new tip and pipette 10ul of gRNA in your CT containing competent cells. Eject tip.
new tip and pipette 10ul of Template DNA in your CT containing competent cells. Eject tip.
incubate on ice for 30 min
Incubate the CT mixture tube for XXX30 seconds in 42ºC (108ºF) water

Recovery
Prepare LB Only media by adding 1.5mL of room temperature water to one of the LB media microcentrifuge tubes and shake to dissolve the LB.
new tip and pipette 1.5mL of LB Only media in your CT containing competent cells and CRISPR reagent. Eject tip.
Incubate the CT tube in normal incubator temp for EC for 2 to 4 hours. This can be extended to 12 hours.

Bring 2 cold LB Strep/Kan/Arab Agar plate to room temperature if needed. Bring cold LB Agar plate to room temperature if needed.


Main Experiment. Using the pipette, add 200uL of your CRISPR transformation mixture on one LB Strep/Kan/Arab Agar plate. Using an cell spreader, gently spread the bacteria around the Strep/Kan/Arab Agar plate and let dry for 10 minutes before putting the lid back on.
Incubate normally for EC. Bacterial growth means success. Failure usually means the bacteria was dead or antibiotic too strong.

Negative Test. Scrape a full innoculation loop off the original DH5A? plate
Using an cell spreader, gently spread the bacteria on the LB Strep/Kan/Arab Agar plate.
Incubate normally for EC. Bacterial growth means FAILURE. Failure means the antibiotic was not good.