Team:Northwestern/Protocols/GFPAssay

From 2012.igem.org

Fluorescence Assay

Procedure

  1. Put 5 mL of LB into new labeled overnight tubes. Remember to make 2 overnight tubes of each (1 for testing OD's in the spectrophotometer and 1 for actually using in the assay).
  2. Take the overnights out of the incubator from the previous night.
  3. Take 100 uL of the overnights from the incubator and place in the new correctly labeled overnight tubes (100 uL of the overnights goes in each copy of each tube).
  4. Grow up the new overnight tubes in the shaker for about 1.5 hours.
  5. While waiting on the cells to grow up, make LB that is at the correct pH for the assay.
  6. Take 4 tubes, label them LB pH7, LB pH4, LB pH3, LB pH2. Put 5 mL of LB into each tube.
  7. Go to the pH probe. Follow the instructions on how to use the pH probe. Check the pH of the solution and slowly add HCl or NaOH to adjust pH. I suggest using .1M NaOH to make the pH7 LB and 1M HCL to make the other pH LBs.
  8. When you are done put the LB in the cold room until you are ready to setup the assay.
  9. Check the OD's of the overnight tubes from the test overnights. If the cells are not at an OD of 0.3-0.4 put them back into the incubator and check again in 15-30 minutes. The goal is to get the cells in log phase which is around 0.4 OD. However, make sure to not overgrow the cells. Remember that it will take approximately an hour to place all the samples into the 96 well plate so I would suggest taking all of the overnights out near 0.3 rather than 0.4.
  10. Once you have all of the cells at about 0.3 OD and the correct pH LB, grab a 96 well plate and start heating up the plate reader (this takes about a half hour to heat up).
  11. To the blank wells just add the correct pH of LB, nothing else. So add 200 uL of LB.
  12. To all of the other wells add 196 uL of the correct pH LB and then 4 uL of the cells you grew up in log phase.

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