Making competent cells
Chemically Competent Cells
- Streak cells onto LB overnight
- grow single colony in 15mL LB overnight
- take 1mL of overnight culture and Grow cells to OD=0.5 in 125mL of LB shaken for 4hrs-ish at 37C 200rpm 20mm orbit in a 250mL erlenmeyer flask with foam plug
- spin down at 3500rpm for 5 min in 8 15mL tubes full up.
- Decant completely, remove all the excess liquid with a pipette (very important to do so but do not disturb the pellet).
6-8 should be done on ice and done as quickly as possible:
- Resuspend one of the 8 tubes with 1mL cold LB
- take the entire resuspension into another spun down tube
- use the liquid from the previous resuspension to resuspend the second tube
- Repeat until all the pellets are resuspended and concentrated into one tube. Resulting suspension should be about 1.2mL. Keep all tubes in contact with ice while resuspending.
- Add that volume of ice cold TSS 2x to the tube and mix gently by pipetting up and down.
- Dispense 100uL into sterile prechilled pcr tube. Makes about 24 reactions worth.
Can potentially be stored at -20C for about 3 weeks with just enough transformation efficiency to be useful. Source.
- 5g PEG 8000
- 1.5 mL 1M MgCl2 (or 0.30g MgCl2*6H20)
- 2.5 mL DMSO
- Add LB to 50 mL