Purification and characterization of EGFP by recombinant Escherichia coli Barney05/10/19

Kan R P T7lac NdeIBglIIXhoI LacI P CMV EGFP XhoIBglII Kan R P T7lac LacI pEGFP-C1 EGFP pET30b(+) Polymerase chain reaction ligation

Chemical transformation 1. Make the competent cell Cultivate over-night E. Coli. cultivate in 3 ml LB MagnificationE. Coli. magnified in 30 ml LB Shake for about 1 hr Centrifuge for 5 mins, 500rpm Discard the super Suspend with CaCl 2 Centrifuge for 5 mins, 500rpm Discard the super Suspend with CaCl 2, then add 50% glycerol Allot to the micro-tube 200  l

(cont’d) 2. Transfer Kan R P T7lac LacI Mix together and then place in the ice for 30 mins Treat with 42 ℃ for 2 mins Treat with the ice-bath again for 5 mins Cultivate with 1 ml LB Shake for 1 hr Spread on the culture medium Cultivate for 12~16 hrs TOP 10

Cultivation Pick any 12 dots randomly Screen Cultivate for 12~16 hrs Colony PCR Extract plasmid DNA Transform into competent cell of BL21(DE3) Cultivate for 12~16 hrs Magnification E. Coli. magnified in 30 ml LB Pick up one line to cultivate in 1 ml LB

(cont’d) To induce EGFP to be made IPTG (0.1mM) React with 2~3 hrs Centrifuge for 5 mins, 500rpm Break with supersonic Suspend with PB buffer Purify with IMAC NEXT PAGE

IMAC ( Immobilize Metal Affinity Chromotography) H2OH2OStarting bufferTarget proteinElution bufferStarting buffer Purified EGFP EDTAH2OH2O recovery

Western-Blot analysis milk 1 st 2 nd NEXT PAGE In Caps Buffer Wash by TBST Buffer Blocking Buffer Anti-polyHistidineAnti-mouse IgG alkaline phosphates-conjugate

(cont’d) 2 nd Wash by TBST Buffer NBT stock solution BCIP stock solution in Alkaline assay buffer