{% extends "layout.html" %} {% block title %}Experiments{% endblock %} {% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %} {% block page_content %}
Aim: To extract Tau plasmid from E. coli (BL21 strain) cells
Materials Required: HiMedia Miller Luria Bertani Agar, HiMedia Miller Luria Bertani Broth, E. coli BL21 cells containing Tau plasmid, Invitrogen's Plasmid Mini-Prep Kit, Kanamycin
Tau plasmid was isolated from BL21 cells by following the protocol provided in Invitrogen’s Plasmid Mini-Prep Kit. The protocol has been provided here for reference.
Aim: Preparation of chemically competent bacterial cells
Materials Required: HiMedia Miller Luria Bertani Agar, HiMedia Miller Luria Bertani Broth, RbCl, MnCl2.4H2O, CH3COOK, CaCl2, glycerol, MOPS, 0.45µ filter
Chemicals | Concentration in the buffer | Amount in the buffer (100ml) |
---|---|---|
RbCl | 100mM | 1.208g |
MnCl2·4H2O | 50mM | 0.990g |
CH3COOK | 30mM | 0.294g |
CaCl2 | 10mM | 0.110g |
Glycerol | 15% | 15ml |
Chemicals | Concentration in the buffer | Amount in the buffer (100ml) |
---|---|---|
PIPES | 10mM | 42mg |
RbCl | 10mM | 24.2mg |
CaCl2 | 75mM | 0.166g |
Glycerol | 15% | 3ml |
Aim: Transformation of BL21 cells with tau plasmid
Materials Required: HiMedia Miller Luria Bertani Agar, Kanamycin (100mg/ml)
Follow the protocol for competent cell preparation
Aim: To induce expression of tau protein in tau plasmid containing BL21 cells and purify tau protein produced
Materials Required: LB medium, 100mg/ml kanamycin, NaH2PO4, Na2HPO4, Isopropyl ß-D-1-thiogalactopyranoside (IPTG), Ethylenediaminetetraacetic acid (EDTA), Beta-mercaptoethanol (BME), NaCl, Phenylmethylsulfonyl fluoride (PMSF), DNase, MgCl2, imidazole, NaOH solution, 0.45µ filter
Carry out the following steps in a laminar air flow under sterile conditions
Carry out the following steps in a laminar air flow under sterile conditions
Components | Concentration in the buffer |
---|---|
NaH2PO4 | 24.59mM |
Na2HPO4 | 75.41mM |
Components | Concentration in the buffer |
---|---|
NaP | 50mM |
BME | 5mM |
EDTA | 1mM |
NaCl | 150mM |
PMSF | 1 |
Components | Concentration in the buffer |
---|---|
NaP | 50mM |
BME | 5mM |
NaCl | 150mM |
Aim: To visualise and identify tau protein by running SDS PAGE
Materials Required: 30% acrylamide mix, 1.5M Tris pH 8.8, 1.0M Tris pH 6.8, SDS, Ammonium persulphate (APS) , N,N,N′,N′ -Tetramethylethylenediamine (TEMED), Tris-HCL, glycerol, (Betamercaptoethanol) BME, bromophenol blue, isopropyl alcohol, Bio-Rad Mini PROTEAN Tetra Cell, Bio-Rad PowerPac system
Components | Volume |
---|---|
dH2O | 1.9 mL |
30% Acrylamide Mix | 1.7 mL |
1.5M Tris pH 8.8 | 1.3 mL |
10% SDS | 0.050 mL |
10% APS (Freshly Prepared Stock) | 0.050 mL |
TEMED | 0.002 mL |
Components | Volume |
---|---|
dH2O | 1.4 mL |
30% Acrylamide Mix | 0.33 mL |
1.0M Tris pH 8.8 | 0.25 mL |
10% SDS | 0.020 mL |
10% APS (Freshly Prepared Stock) | 0.020 mL |
TEMED | 0.002 mL |
Components | Amount |
---|---|
Tris-HCl | 1 mL |
SDS | 0.2 g |
Glycerol | 1 mL |
BME | 0.5 mL |
Bromophenol | 0.001 g |
Aim: To determine the concentration of protein extracted and purified using Bradford Assay
Materials Required: Bovine serum albumin (BSA), Bradford reagent
Concentration (µg/ml) | Volume of BSA solution | Volume of distilled water(µl) |
---|---|---|
100 | 100µl of stock soln. | 900 |
80 | 800µl of soln. 1 | 200 |
60 | 750µl of soln. 2 | 250 |
40 | 667µl of soln. 3 | 333 |
20 | 500µl of soln. 4 | 500 |
10 | 500µl of soln. 5 | 500 |
Aim: To phosphorylate purified tau protein
Note: The following protocol has been optimised for 2.86mM (as measured by Bradford Assay) of purified tau protein
Materials Required: Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), 50mM MgCl2 solution, 10mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES buffer), Promega’s r-ATP (10mM), GSK-3β enzyme, Laemmli buffer
Components | Concentration (mM) | Amount |
---|---|---|
EGTA | 0.625 | 0.0002375g |
MgCl₂ soln. (50mM) | 0.6 | 12µl |
HEPES (10mM) | 5 | 500µl |
H₂O | - | 282µl |
ATP (10mM) | 0.4 | 4µl |
GSK-3β | - | 2µl |
Aim: Click reaction of C8-Alkyne dCTP with [S, R, S]-AHPC-PEG1-Azide (VHL Ligand)
Materials Required: HEPES Buffer (10 mM, pH 7.8), CuSO4, Sodium L-ascorbate, C8-Alkyne dCTP, [S, R, S]-AHPC-PEG1-Azide
Aim: To verify aptamer binding to 231-phosphorylated tau by performing an electrophoretic mobility shift assay
Materials Required: 30% Acrylamide solution, Tris-HCl, 10% APS solution, TEMED, Phospho-Tau (7ng/ml in Laemmli buffer and phosphorylation buffer, DNA aptamer, nuclease free water, polyvinylidene fluoride (PVDF) membrane, Semi-Dry Transfer Cell (Tran Blot SD BioRad), TBE Buffer, TBS Buffer, skimmed milk powder, Anti-231pTau Ab (Rabbit monoclonal from ThermoFisher), Anti-Rabbit HRP-conjugated Ab, X-ray plates, ECL substrate, 5M NaCl, 1M KCl, 1M MgCl2, 0.5M EDTA, 1M DTT, 10mg/ml BSA, Laemmli Buffer, Ponceau stain (optional)
Components | Volume (in µl) |
---|---|
1M Tris-HCl pH 7.5 | 50 |
5M NaCl | 10 |
1M KCl | 200 |
1M MgCl2 | 5 |
0.5M EDTA pH 8.0 | 10 |
1M DTT | 5 |
10mg/ml BSA | 25 |
ddH2O | 695 |
Components | Volume (in µl) |
---|---|
4X Binding Buffer | 5 |
Phosphorylated Tau Protein | 10 |
DNA Aptamer | 1 |
Nuclease Free Water | 4 |