Molecular Pathology Unit & Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts 02129
Use an Eppendorf repeating pipettor and a sterile 12.5-ml dispenser tip to aliquot 1 ml of bacterial growth medium (e.g., LB) into the wells of a Greiner MasterBlock (Model #780271) or a Corning Costar 2-ml Assay Block (#3960).
Use a pipette tip to lightly touch a colony, submerge it in the well to be inoculated and briefly pipette up and down. Discard the tip.
Cover the block with a Qiagen Airpore strip (#19571). Seal all wells tightly.
Place the block in the Microtitertron (Appropriate Technical Resources). Check carboy to make sure that there is enough water, and set the humidity to 80%. Set shaker to 900 rpm and 37°C.
The following morning, use the repeating pipettor to transfer 1 ml of prewarmed medium to wells in a 96-well block. Use a multichannel pipettor to transfer 25 μl of the overnight culture to the 1 ml of fresh medium. Place in Microtitertron with the same settings as the overnight culture except that no Airpore strip is needed.
-
To monitor growth of the subcultures, use a multichannel pipette to transfer 200 ul of culture to a nonsterile 96-well plate and take an OD595 reading on a Bio-Rad Microplate Reader 680.
The absorbance should fall within the linear range of the plate reader (determined empirically for each machine). Note that the subculture volume is sufficient to allow for at least three checks of the OD595.
If the cultures are not growing at the same rate, keep them in the 96-well plate at room temperature since they will not grow appreciably at that temperature. Just pipette up and down to agitate the cells, and then reread the OD595 when all cultures are ready. Alternatively, if some cultures are severely lagging, lyse the cells that are ready and then keep them at room temperature while waiting for the others. When finished with the Microtitertron, do not forget to turn off the humidity.
-
Prepare lysis master mix solution: 10 μl of Popculture (Novagen #71092) + 1 μl of R-Lysozyme (Novagen #71110) (4 units/μl) for each assay to be performed.
The stock of lysozyme that I have prepared is at a concentration of 400 units/μl. Dilute the stock back to 4 units/μl with sterile Dilution Buffer. Diluted lysozyme can be used only on the day it is prepared. Discard the remainder.
Use a repeating pipettor to aliquot 11 μl of lysis master mix into the wells of a nonsterile 96-well polystyrene plate.
Set up the kinetic protocol to take readings as follows:
OD415
28°C
Absorbance range: 0.1–1.0
Time limit of 3600 seconds (1 hour)
-
Use the mix function on the M8 to transfer 100 μl of culture from the wells (where the OD595 was read) to the wells that contain the lysis buffer. Mix up and down. Leave at room temperature for at least 15 minutes to ensure complete lysis.
Lysed cells are stable for at least 18 hours at room temperature.
Z buffer (see recipe at end of the protocol) may be kept at room temperature without the β-mercaptoethanol (β-ME). Before doing an assay, prepare fresh Z buffer with β-ME (use 2.7 μl of β-ME for every 1 ml of Z buffer).
Remember to perform two assays for each subculture. In a solution tray, mix together sufficient Z buffer (containing β-ME) and ONPG for performing assays; use 135 μl of Z buffer with β-ME and 30 μl of ONPG (4 mg/ml dissolved in H2O and stored at –20°C) FOR EACH ASSAY that will be run. Use the M8 to aliquot 165 μl of this Z buffer/β-ME/ONPG mixture into the designated reaction wells.
Transfer 15 μl of lysed cells to each reaction well using a multichannel pipettor. Move the plate to the plate reader and start the assay.
Calculate Miller units as described in Thibodeau et al. (Biotechniques 2004).
