Transfomation for protein expression

Creating Transfomation for protein expression

• 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