User:Daniel Goodman/Notebook/Cluzel/2010/03/03

Glycerol Stocks for MG1655 strains

 * 2 culture tubes
 * 4 ml LB in each
 * 4 ul of Amp in one (for GFP strain)
 * took one average-sized colony from each
 * put in 37°C shaker (at ~12:30 pm)

Forgot to take out the cultures on Friday, so doing this again today.

Reminder: Take culture tubes out of shaker at ~6:30 and put stocks in glycerol! (done)

Agarose

 * making 5% agarose (.3 g in 6 mL) for molds that are easier to manipulate and less likely to fracture/distort

Experiment
I get the stage at ~1:30. I will pour 2 featureless molds and try transferring again, gently, to see if we get a clear spot boundary upon transfer.


 * Put 5% agarose in at 12:56
 * Pour molds into PDMS at 1:15
 * Molds will dry at 1:35

''Result: 5% agarose is extremely bright in GFP field, but no cells were seen in phase contrast. Perhaps more 'wetness' is required to transfer cells.''

Note: see below: 5% agarose seems to work fine, no idea why GFP field was so bright before; probably bad settings on the scope

Experiment 2
Spot cells directly on gel surface with crimped 30 gauge needle dipped in high-OD cell solution. Trying with both 5% and 3% agarose. Spot cells on slide (w/ needle). Trying with both 3% and 5% agarose. Using GFP cells grown up earlier today.

Results

 * Did not find cells on Direct application 3%
 * Did find cells on Direct application 5%, located in one region (with some separation outward, and a 'canyon' where the tip ground into the agarose)
 * It is unclear if this is due to my hand movement or spread of the cells after application
 * Because I used serrated pliers to crimp the needle head, there is a serrated pattern in the agarose where the cells landed; could we stamp the pattern and cells onto blank agarose at the same time?
 * Found very few cells on Xfer 5%, but was pressed for time.

It is generally hard to find cells if they are only in one location. Features are difficult to locate and so it is difficult to keep the agarose surface in focus. It took me about 10 minutes to find the cell patch on 5% direct application the first time, and another 45 minutes to locate it again!

Images are saved as 3-3* in the ~/dbg dir on the camera computer.


 * }