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Team:UCL/Project/Protocols
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Revision as of 11:34, 26 September 2013
Bacterial Lab Protocols
1.4% Agar
In 500mL Duran bottle insert:
| Reagent | Required Quantity |
|---|---|
| Agar | 7g |
| RO H2O | 500mL |
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 |
0.1M CaCl2/15% glycerol
In a 50mL Falcon insert:
| Reagent | Required Quantity (mL) |
|---|---|
| 1M CaCl2 | 5 |
| 100% Glycerol | 7.5 |
| RO H2O | 37.5 |
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 |
LB Media
In 500mL Duran bottle insert:
| Reagent | Required Quantity |
|---|---|
| LB Broth | 10g |
| RO H2O | 500 mL |
Generating Competence Cells
Locate a glycerol stock of untransformed E. coli, streak cells onto minimal agar plates and incubate at 37C for 16 hours.
Once complete, pick a colony from the plate and place into a 50mL Falcon, containing 5mL LB & 100 uL 1M MgSO4 for 16 hours.
After this, inoculate a 100mL shake flask with 1mL of culture from the Falcon tube. Take absorbance readings every 30 minutes until the absorbance reading is above 0.3. Once this is achieved, transfer the contents into two 50mL Falcon tubes and place on ice for 10 minutes. Perform centrifugation (~6,000 RPM) for 5 minutes and then resuspend in 10mL Calcium Chloride. Aliquot into eppendorf tubes (~500 uL per tube) and then store at very low temperatures (<-50C).
Streaking Plates
Obtain agar plates (as many as required), streaking loops and cells to be streaked. Dip a streaking loop in the cell culture, and gently (so there is no damage to the agar) streak the loop onto the plate as described in the diagram below. Once finished, incubate at 37C overnight.
Plate generation (AMP, CMP & NoDrug)
Heat up 50 mL of agar until molten (usually ~300 seconds using a 800W microwave). Douse in cold water to lower temperature. When still warm, but able to handle, it is possible to add an antibiotic drug for selection purposes (~50 uL). Once this complete, pour ~10mL into a petri dish and ensure that the whole surface is covered. Leave lid off for 30 minutes. Place lid on dish and then use, or store at ~5C.
Streaking Plates
Obtain agar plates (as many as required), streaking loops and cells to be streaked. Dip a streaking loop in the cell culture, and gently (so there is no damage to the agar) streak the loop onto the plate as described in the diagram below. Once finished, incubate at 37C overnight.
50X to 1X Dilution
To a 1L Duran bottle, insert:
| Reagent | Required Quantity (mL) |
|---|---|
| 50X TAE Buffer | 20 |
| RO H2O | 980 |
Mammalian Lab Protocols
Passaging Adherent Cells
In order to keep cells healthy or increase stock, they must be sub-cultured - moving some cells from a previous culture into a new container with fresh growth medium. Here, we assume a 100mm dish. All solutions/equipment that come in contact with the cells must be sterile and work must be done in a laminar flow hood.
1) Pipette spent medium and discard to waste.
2) Gently wash cells with PBS (5-10mL), then remove PBS to waste. Be careful not to disturb the cellular monolayer. This removes serum residue with trypsin inhibitors.
3) Add trypsin (2-5mL) to suspend cells. Ensure monolayer is covered. Incubate for 3-5 minutes at 37C.
NOTE: Care should be taken to avoid leaving cells exposed to the trypsin longerthan necessary. Care should also be taken that the cells not be forced to detach prematurely, as this may result in clumping.
4)Add serum-containing medium(10mL) and pipette the cells up and down until the cells are dispersed into a single cells suspension.
5) Add the appropriate volume of cell suspension (dependent on confluence/cell count - generally for 100% confluence split 1:4) to a new flask/dish containing medium (end volume 10mL).
7) Place dish(es) in incubator at 37C. Leave for 3-4 days before next passage.
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 for gene expression.
