# Team:UCL/Project/Protocols

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Streaking Plates

Streaking Plates

## Revision as of 10:11, 26 September 2013

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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

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.

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Mammalian Lab Protocols

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.