User:Daniel Goodman/Notebook/Cluzel/2010/02/26

Cluzel Lab Notebook Daniel Goodman

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Glycerol Stocks for MG1655 strains Forgot to take cultures out on Friday, so doing this again. 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 12:15 pm) Retry molds again Will pour 2 molds using silicon chips to generate features, so it will be easier to focus. This must done in the humidity and temp. controlled room so features generate nicely. I am worried about desiccation of the agarose, so I'm going to keep it in the Humidity and Temp controlled room (HTC room) for as long as possible, and get the imaging equipment ready to see if the gel still distorts like on Wednesday. Jeff thinks that using the cover slip on top might not be necessary, but as long as it is placed on top carefully it should not 'squish' the features and it will help reduce moisture loss once the device is out of a high-humidity environment by limiting exposed surface area. There will be no exposed surface area in the actual device, so this hopefully will not be a problem. Make agarose as normal: 5 mL LB, 0.150 mg agarose powder. Vortex in tube, heat at 80°C for 15 minutes. (put in at 12:30 - bath was not on; took out at 1:30) Clean chips in DI water bath (80°C) in flask for 5-10 minutes. (put in at 12:32 - bath was not on; took out at 1:30) After agarose is hot, pour into mold in the HTC room, wait 1 hour for features to form. (put in at 1:37) Put cells on glass slide right before agarose is done setting in normal conditions, move into HTC room and remove from mold and put on top of cells immediately. Cover with square cover slip to maintain moisture. Check spot size before agarose application Spotted 5 0.2 uL dots of stationary phase cells onto a glass slide, imaged to check spot diameter. On the order of 0.5 to 1.5 CCD chips in spot diameter, if correctly applied. Spots dry within 1 minute. Salt crystals formed in dried spots. (Images saved in C:\MM\images\dbg\expt* dir on camera computer) Compare spot size before and after agarose application Prep cells to remove salt crystalization and dilute Spun .1 mL of stationary phase culture down, removed supernatant, added 1 mL DI water. Cells are unhappy, but easier to see. Spotting on slide Spotted 2 0.2 uL dots of stationary phase cells onto a glass slide. Wait 2 minutes to dry. Do this for two different slides. Moved slides into HTC room, removed agarose from mold and placed agarose on top of one of the two cell spots. Placed cover slip on top. Quickly moved slides to scope and imaged, one at a time (kept the other in the HTC room to retain moisture for longer). Cells remain on agarose, with the majority constrained to one region (little or no 'wicking' observed). (Images saved in C:\MM\images\dbg\expt3* dir on camera computer) Transfer from slide to slide Used non-spun high-OD cells. Spotted one small dot (0.2 uL) on glass slide, wait to dry. Removed agarose from mold, placed agarose on the spot, picked up agarose, moved to new spot. Do the cells transfer well? Is there a clear transfer boundary? Agarose was deformed during transfer process, need to be more careful or perhaps have a more gentle way of performing transfer. However, cells transferred successfully, albeit at lower concentration. Hard to see localization, due to breakup and distortion of gel. Images saved in C:\MM\images\dbg\expt4*