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Team:UCL/Project/Protocols
From 2013.igem.org
Bacterial Lab Protocols
5X M9 Salts
In 500mL Duran bottle insert:
| Reagent | Required Quantity |
|---|---|
| Na2HPO4 | 32g |
| KH2PO4 | 7.5g |
| NaCl | 1.25g |
| NH4Cl | 2.5g |
| RO HCL | 500ml |
Minimal Agar
Mix:
| Reagent | Required Quantity |
|---|---|
| 5x M9 Salts | 10mL |
| 2 mg/ml Thiamine | 50µl |
| 20% D Glucose | 1 mL |
| 1M CaCl2 | 5µl |
| 1M MgSO4 | 100µl |
| 1.4% Agar | 39 mL |
1.4% Agar
In 500mL Duran bottle insert:
| Reagent | Required Quantity |
|---|---|
| Agar | 7g |
| RO H2O | 500mL |
Mammalian Lab Protocols
Stable Transfection Of Adherent Cells
For the stable transfection of eukaryotic adherent cell types in a single well of a 6-well plate. When transfecting multiple wells, make a 'master mix' with 110% of all solutions.
1) The day before transfection, seed 0.9-4x10^5 cell per well of the six well plate with 2ml of appropriate growth medium. This should produce a confluence of 40-80% for the next day's transfection.
2) Incubate cells in their normal growth conditions (37^0 C and 5% CO2)for 24 hours.
3) Dilute 2µg of DNA dissolved in TE buffer (min conc. 0.1µg/µl) with serum, protein and antibiotic free medium (to avoid macromolecular interference with complex formation) to a total of 100µl. Mix.
4) Add 10µl of superfect (SF) reagent to the solution. Vortex for 10 seconds.
5) Incubate at room temperature for 5-10mins to allow for complex formation.
6) Meanwhile, gently aspirate growth medium from dish and wash cells with 3ml.
7) Add 600µl of cell growth medium (with serum and antibiotics)to reaction tube. Mix up and down with pipette and immediately transfer total volume to well.
8) Change medium and wash with PBS.
9) Incubate for 24-48 hours.
9) Assay fro gene expression.
