Purification and characterization of EGFP by recombinant Escherichia coli

1. Purification and characterization of EGFP by recombinant Escherichia coli Barney 05/10/19

2. BglII XhoI EGFP NdeI BglII XhoI PCMV pEGFP-C1 PT7lac pET30b(+) Polymerase chain reaction KanR LacI EGFP ligation EGFP PT7lac KanR LacI

3. Chemical transformation 1. Make the competent cell Shake for about 1 hr E. Coli. cultivate in 3 ml LB Cultivate Magnification E. Coli. magnified in 30 ml LB over-night Centrifuge for 5 mins, 500rpm Discard the super Suspend with CaCl2, then add 50% glycerol Centrifuge for 5 mins, 500rpm Allot to the micro-tube Discard the super Suspend with CaCl2 200 l

4. PT7lac KanR LacI (cont’d) 2. Transfer TOP 10 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 Cultivate for 12~16 hrs Spread on the culture medium Shake for 1 hr

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

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

7. H2O Starting buffer Target protein Elution buffer Starting buffer EDTA H2O IMAC (Immobilize Metal Affinity Chromotography) recovery Purified EGFP

8. 1st 2nd Western-Blot analysis In Caps Buffer Wash by TBST Buffer milk Blocking Buffer Wash by TBST Buffer NEXT PAGE Anti-mouse IgG alkaline phosphates-conjugate Anti-polyHistidine

9. 2nd (cont’d) Wash by TBST Buffer NBT stock solution BCIP stock solution in Alkaline assay buffer