purification and characterization of egfp by recombinant escherichia coli barney05/10/19
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Purification and characterization of EGFP Purification and characterization of EGFP by recombinant by recombinant Escherichia coliEscherichia coli
BarneyBarney
05/10/1905/10/19
KanR
PT7lac
NdeI BglII XhoI
LacI
PCMV
EGFP
XhoIBglII
KanR
PT7lac
LacI
pEGFP-C1
EGFP
EGFP
pET30b(+) Polymerase chain reaction
ligation
Chemical transformationChemical transformation 1. Make the competent cell 1. Make the competent cell
Cultivateover-night
E. Coli. cultivate in 3 ml LB
Magnification E. Coli. magnified in 30 ml LB
Shake for about 1 hr
Centrifuge for 5 mins, 500rpm
Discard the super
Suspend with CaCl2
Centrifuge for 5 mins, 500rpm
Discard the super
Suspend with CaCl2, then add 50% glycerol
Allot to the micro-tube
200 l
(cont’d) (cont’d) 2. Transfer2. Transfer
KanR
PT7lac
LacI
Mix together and then place in the ice for 30 mins
Treat with 42℃ for 2 mins
Treat with the ice-bath againfor 5 mins
Cultivate with 1 ml LB
Shake for 1 hr
Spread on the culture medium
Cultivate for 12~16 hrs
TOP 10
CultivationCultivation
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)(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 withPB buffer
Purify with IMAC NEXT PAGE
IMAC (IMAC (Immobilize Metal Affinity Chromotography)Immobilize Metal Affinity Chromotography)
H2O Starting buffer Target protein
Elution buffer
Starting buffer
Purified EGFP
EDTAH2O
recovery
Western-Blot analysisWestern-Blot analysis
milk
1st
2nd
NEXT PAGE
In Caps Buffer
Wash by TBST Buffer
Wash by TBST Buffer
Blocking Buffer
Anti-polyHistidineAnti-mouse IgG alkaline phosphates-conjugate
(cont’d)(cont’d)
2nd
Wash by TBST Buffer
NBT stock solution
BCIP stock solution
in Alkaline assay buffer
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