- Prepared 2 ON cultures (37C without shaking) in incubator (5ML TSB + RN4220 Staph Aureus)
- To be taken out at 1030am Friday (July 30th)
Growth Curve Data From July 28th, 2010
- Strain: H1001 (dAGRD staph strain from IOWA)
- Media: B2 Broth
- Culture:Strain Dilutions: 1:300 (50ul:15ml), 1:400 (37.5ul:15mL)
Growth Curve Experiment
|Time|| Control || 1:300 || 1:400 ||
Growth Conditions: 200rpm, aerated at 37C
- 2 colonies grew for construct P2 promoter + E00840. At least 3 colonies grew for every other construct (combination of P2, J23100, Pkat, Pbad and E00840, I13504).
- PCR screen 2 or 3 colonies from each construct.
Table 3. Master Mix A
|Reagent||1X rxn volume (uL)||Master Mix|
|10x Thermol Pol Buff||2.5||x25||62.5|
|10uM FW primer||1.25||x25||31.25|
|10uM RE primer||1.25||x25||31.25/td>|
- Ran PCR using UBC iGEM screening program (25 cycles).
- Ran a gel using 1% agarose and 0.5% TBE buffer solution over 50 minutes. Loaded 100 bp NEB QuickLoad ladders (contains reference bands for 100 bp-1000 bp at 100 bp intervals and also 1200 and 1536 bp).
- Poor band resolution in the ladders (poor separation between ~700 bp to ~1536 bp). Difficult to tell if bands were in the correct place (~1250-1350 bp). Different colonies from the same plate (containing supposedly the same construct) sometimes had slight differences in apparent band location. Calvin Chan suggests higher concentration of agarose and longer run times. Also, initally run at 30 V, allowing loading marker to move into the gel from the wells before ramping up the voltage; this should let all bands start moving through the agaorse at ~ the same time, instead of slight differences due to location of DNA in the well initially.
- Decided to inoculate overnight cultures from index plate if bands were roughly in the right expected location (around the highest reference band). If 2 of the bands were slightly different, inoculated 2 colonies. Growth medium = LB + Chlorophenicol (25 mg/mL). Used Lagally lab incubator (220 rpm, 37). Start time for incubation: 6:30 pm.