July 1st
Re-streak of:
His-TEV-protein plasmid (provided by professor LG Mårtensson), clone 1-2
BBa_K525998 (promotor+RBS), clone 1-4
New transformation of:
RFP PSB1C3-J06504 again since the bacterias did not grow.
July 2nd
Re-streak of:
RFP PSB1C3-J06504
Transformation of:
RFP-control with electrocompetent cells
Over-night of:
PSB1C3 – T7 promotor
His-TEV-protein
Preparation of starter:
10 colonies of transformed His-TEV-protein were used. Induction over-night.
July 3rd
Plasmid preparation of:
PSB1C3-T7 promoter, clone 1-3
His-TEV-protein, clone 1, 2
Over-night on:
PSB1C3-J06504-RFP
Re-streak of:
RFP-control
Preparation of:
Large culture of transformed His-TEV-protein.
Measurement of OD600 and induction with IPTG over-night.
July 4th
Plasmid preparation of:
J06504-RFP, clone 1, 2
Transformation of:
PSB1C3-E1010-RFP
Competency control of:
Electro competent cells
Harvest of large culture by centrifugation followed by lysation of bacterial cells through sonication.
Purification of:
His-TEV-protein (lysate from sonication) by the use of nickel-column.
July 5th
Re-streak of: PSB1C3-E1010-RFP, clones 1-6
July 6th
Over-night of: PSB1C3-E1010-RFP, clones 1-6
July 7th
Plasmid preparation of:
PSB1C3-E1010-RFP overnight-culture, clone 5
July 8th
Analysis of protein content in:
Fractions from nickel-column purification by the use of Bradford assay.
Fractions containing protein was analyzed by the use of SDS-page.
July 9th
Restriction digest performed by spin-kit and the use of Xba1 and Spe1 followed by ligation and transformation (electroporation).
July 10th
Fractions containing His-TEV-protein was set on TEV-cleavage over-night.
July 11th
Reversed nickel-column purification of:
His-TEV-protein after TEV cleavage.
Fractions from the reversed nickel-column purification as well as a fraction both before and after TEV cleavage was controlled by SDS-page.
July 15th
Restriction digest test on all restriction enzymes (except FD PstI) and put on agarose elektrofores gel. (EcoRI, XbaI, SpeI, PstI)
Nanodrop of PSB1C3-E1010 and His-Tev to control the purity and concentration.
July 16th
Analyze of the agarose gel including staining with EtBr, destaining with water and visualization under UV-light.
July 21st
New fransformation of PSB1C3-E1010 and His-Tev scince the concentrations from previous plasmid preps were too low.
July 29th
O/N on His-TEV-protein, clones 1-4.
"
