Transfomation for protein expression
Creating Transfomation for protein expression
[edit]- take competent cells (Nico21(DE3)pLysN) from minus 80 freezer
- pipet 100 microliters and mix with 0.5 microliter of given plasmid
- incubate on ice for 30 minutes
- pour some water on the PCT PCR machine and heat up to setting "42-30" and put on it for about 30~45 seconds
- take it out and put on ice, let sit on ice for 2 minutes
- add 750 microliters of SOC
- put in shaker for about two hours
- spin for one minute
- dump out liquid but leave about 50-100 microliters
- suspend bacteria with pipette
- spray on LB/KC plate and spread with glass beads
- put in incubator overnight, agar side up
- ---------> next day
- wash out colonies with LB/KC or LB/AC and pipette into a 50 mL plastic tube
- add LB/KC or LB/AC until it reaches 15 mL
- put in shaker for about 2-3 hours
- add to 1 or 2 Liters of LB/KC or LB/AC
- grow in shaker
- induce with 0.5 mL of 1 M IPTG per bottle
- induce at 37 oC for 4 hours, or room temperature overnight
- spin down cells, combine, weigh cell pellet weight and store cells at -80 freezer