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Team:Linkoping Sweden/Notes
From 2014.igem.org
Here you can find notes giving a detailed insight into what we have attempted in the lab during this summer and fall. Read and enter the exciting Lab World of the LiU iGEM Team of 2014!
June
June 19th
Supercompetent E-Coli was given to us from one of our professors. We tested the health of the bacteria by letting them grow on a control plate at 37 ºC.
June 24th
Transformation of RFP control (10pg/ul) plasmid into supercompetent cells by heat shock method.
June 25th
Did the transformation of RFP control again since the first transformation went wrong.
June 26th
RFP control plate looked good this time.
June 30th
Heat shock transformation of:
His-TEV-protein plasmid (provided by professor LG Mårtensson)
BBa_K525998 (promotor+RBS)
RFP PSB1C3-J06504
July
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.
August
This month we didn’t do much work in the laboratory since we focused more on human practice.
September
September 8th
Plasmid preparation on epitope sequence and RFP PSB1C3-E1010.
Restriction digest on epitope, RFP PSB1C3-E1010 and backbone. The epitope was cleaved with EcoRI + SpeI, RFP PSB1C3-J06504 with XbaI + PstI and the backbone with EcoRI + PstI.
Clean-up on the digest products.
September 9th
Ligation of epitope sequence, RFP PSB1C3-E1010 and linearized backbone (according to protocol from Thermo Scientific).
Control of digest and ligation on agarosegel.
September 10th
PCR with primers on His-TEV.
Clean-up on His-TEV.
Transformation of epitope + RFP (E1010).
September 11th
Re-streak of epitope + RFP (E1010).
September 12th
Over-night culture of epitope + RFP (E1010).
September 15th
Plasmid preparation and glycerol stock on epitope + RFP (E1010).
Digest on His-TEV and RFP backbone (PSB1C3-J06504).
Clean-up on His-TEV and RFP backbone (PSB1C3-J06504).
Gel extraction kit on RFP backbone (PSB1C3-J06504) (to purify the backbone).
Ligation of His-TEV + backbone.
September 17th
Transformation of His-TEV + backbone.
Plasmid preparation and glycerol stock of pUC-plasmid.
September 18th
Plasmid preparation on RFP backbone (E1010) and RFP backbone (J06504).
Re-streak of His-TEV + backbone.
September 22nd
Digest of RFP backbone (J06504) with SpeI and EcoRI.
Clean-up on RFP backbone (J06504).
Gel extraction kit on backbone.
Ligation with purified backbone and His-TEV.
Over-night culture with RFP (J06504).
RFP (J06504) from glycerol stock was put on agar plate with chloramphenicol resistance.
September 23rd
Agarose gel with His-TEV after ligation.
Plasmid preparation on RFP (E1010), RFP (J06504) + Epitope and RFP (E1010) + Epitope.
Cultivation on agar plate with RFP (J06504) from glycerol stock.
September 24th
Over-night culture with RFP (J06504).
September 25th
PCR on RFP (E1010) + epitope and RFP (J06504) + epitope.
Transformation of His-TEV.
September 29th
Digest on RFP (E1010) + pUC plasmid with epitope x5.
Digest on RFP (E1010) + pUC plasmid with epitope x5 + backbone.
Digest on RFP (J06504) + pUC plasmid with epitope x5 + backbone.
Clean-up on all three.
Ligation on all three.
Transformation of all three.
September 30th
Colony-screening on all three products from September 29th.
October
October 1st
Digest on RFP (E1010) + pUC plasmid with epitope 2.
Digest on RFP (E1010) + pUC plasmid with epitope 2 + backbone.
Digest on Digest + pUC plasmid with epitope 2 + backbone.
Clean-up on all three.
Ligation on all three.
PCR-colony screening on all products from the clean-up and ligation.
October 2nd
All six products from October 1st was run on agarose gel.
PCR on the His-TEV sequence.
October 3rd
New agarose gel for the six products from October 1st since the last one gave bad results.
Digest, clean-up and ligation on His-TEV.
Transformation of His-TEV.
The digest, clean-up and ligation products of His-TEV was run on agarose gel.
October 4th
PCR colony screening on His-TEV, RFP (E1010) + epitope x5, RFP (J06504) + epitope x5, RFP (E1010) + epitope 2 and RFP (J06504) + epitope 2.
The PCR samples was loaded on an agarose gel.
Over-night culture on RFP (E1010) + epitope x5, RFP (J06504) + epitope x5, RFP (E1010) + epitope 2 and RFP (J06504) + epitope 2.
Re-streak on the His-TEV transformation from October 3rd.
October 5th
Over-night culture on His-TEV.
October 6th
Plasmid preparation on on His-TEV, RFP (E1010) + epitope x5, RFP (J06504) + epitope x5, RFP (E1010) + epitope 2 and RFP (J06504) + epitope 2.
October 8th
Made starter on RFP (E1010) + epitope 2 and RFP (E1010) + epitope x5.
October 9th
Cultivation on RFP (E1010) + epitope 2 and RFP (E1010) + epitope x5.
October 14th
FITC of monoclonal and polyclonal antibodies (according to protocol from Thermo Scientific).
What we’ve planned to do after the wiki-freeze:
October 18th
Cultivation on clones which are correct from sequensation.
October 19th
Protein expression on all clones from the cultivation.
October 20th
Protein purification.
October 21st
Mix of antibodies and expressed proteins (combination of different epitopes and RFP E1010/RFP J06504).
Purify the antibody/protein mixture.
October 22nd
Test FRET with fluorescence on all mixtures.
