September 1st

Purification of PCR omega fragment

Making gene ruler

Running the purified product

Nanodrop the purified product : 110 ng/μL

Inoculating bacteria with pBluescript KS and pSB1C3 containg alpha fragment

Spreading BL21 and Top10 on agar plates with chloramphenicol antibiotic to check the optimum chloramphenicol concentration.

September 3rd

Nanodrop the large scale isolation product:

pBluesript KS: 69.5 ng/μL

Alpha fragment : 179.5 ng/μL

Restricting pBluescript KS using EcoRv

Nanodrop the restricted plasmid

Making competent cells

September 4th

Testing te chloramphenicol concentration

Testing the enzymatic activity from PstI

Making agar plates with different concentration of chloramphenicol

Running the result of enzymatic activity test

September 5th

Re-testing the enzymatic activity using different buffers

Running

Checking the chloramphenicol test result

Transforming:

Omeg fragment-pBluescript KS

(G4S)4 -pSB1C3 (ligated 3/9)

Lox511-psB1C3 (ligated 3/9)

Lox511-psB1C3 (ligated 25/8)

Lox511-psB1C3 (ligated 25/8)

(G4S)4 -pSB1C3 (ligated 25/8)

pQE80L

Re-ligating omega fragment-pSB1C3

September 6th

Restricting:

Omega fragment + EcoRI

pSB1C3

Omega fragmetn +XbaI

pBluscript KS + XbaI

Alpha fragment + XbaI

Running

Nanodrop the purified digested plasmid

Omega fragment + EcoRI = 7.2 ng/μL

pSB1C3 = 3.8 ng/μL

Omega fragment + XbaI = 7.7 ng/μL

pBluescript KS + XbaI = 9.8 ng/μL

Alpha fragment + Xba I = 0.35 ng/μL

Running

Inoculating bacteria with pSB1C3-alpha fragment in 100mL broth

September 9th

Large scale pSB1C3-alpha fragment isolation

Restricting pBluescript KS using EcoRV

Digesting linearized pSB1C3 and linearized omega fragment using PstI

Making 750mL LB Broth

Making agar plates

Digesting pSB1C3-alpha fragment

Purifying the digested using LMA

Blunting Lox511 and (G4S)4 linker

Running

September 11th

Small scale isolation

Running the small scale isolation product

September 13th

Re-run the small scale iolation product

Inoculate the bacteria containing pSB1C3=omega fragment

September 14th

Running small scale isolation product

PCR Colony

Large scale isolation

Re-ligating omega fragment with pSB1C3

September 15th

Running

Digested pQE+PstI

PQE

Digested alpha fragment+PstI

Alpha fragment

Marker

5

8

9

15

Large scale isolation pBluescript KS-omega

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