Difference between revisions of "20.109(S11):Complete DNA design (Day2)"

Introduction

Protocols

Part 1: Complete DNA design

Part 2: Test liquid cultures for B-gal production

FROM NK, NEEDS REVISION

With this assay you will determine the amount of beta-galactosidase activity associated with a sample of bacterial cells. These cells were engineered to overproduce beta-galactosidase. You will dilute the cells and measure each dilution in duplicate to gain some confidence in the values you measure. A table is included here to help you organize your assay, but you can make one of your own if you prefer.

1. Retrieve 3 ml of the bacterial sample you'll be measuring, called "NB5."
2. In eppendorf tubes, make 1 ml of a 1:10 dilution of NB5, using Zbuffer as the diluent, then use 100 ul of this dilution to make another 1:10, for a final concentration that's 1:100.
3. Transfer 650 μl of the 1:10 dilution to a cuvette and measure the OD600 of this sample. Blank the spectrophotometer with Zbuffer or water. The density of the 1:10 dilution should be in the linear range of the spectrophotometer and your reading can be used to calculate the OD600 of your undiluted cells and your 1:100 dilution.
4. Add 400 μl of Zbuffer to 7 eppendorf tubes labeled 0-6.
5. Add 100 μl of the appropriate cell dilution to each tube. See chart above for guidance. Add 100 ul of Zbuffer to tube 0, to serve as your blank.
6. Next you will lyse the cells by add 20 μl of 0.1% SDS to each eppendorf.
7. To better lyse the cells, you should also add 30 μl of chloroform (CHCl₃) to each tube. Do this in the hood since chloroform is volatile and toxic. You will need to hold the pipet tip close to the eppendorf as you move between the chloroform stock bottle and your eppendorfs since chloroform has a low surface tension and will drip from your pipetmen. Be sure to dispose of your pipet tips in the chloroform waste container located on the right side of the hood.
8. To really really lyse the cells, vortex the tubes for 10 seconds each. You should time these precisely since you want the replicates to be treated as identically as possible.
9. Start the reactions by adding 100 μl of ONPG to each tube at 10 second intervals, including your blank. Invert to mix.
10. Stop the reactions by adding 250 μl of Na₂CO₃ to each tube once sufficient yellow color has developed. “Sufficient” is defined as yellow enough to give a reliable reading in the spectrophotometer, best between 0.3 and 1.0. It is about the amount of yellow color that you see for the yellow tips for your P200. Use today's practice assay to learn what this looks like. Try to stop the duplicate reactions at 10 second intervals and be sure to note the time you are stopping the reactions. Also be sure to remember that adding the Na₂CO₃ makes the reactions more yellow.
11. When all your samples have been stopped, add 250 μl of Na₂CO₃ to the blank and spin all the tubes in the microfuge for 1 minute at 13,000 RPM to pellet any cell debris.
12. Move 0.7 ml of each reaction to plastic cuvettes. Avoid the chloroform that will be at the bottom of your tubes. If you add the chloroform to the cuvettes, it will "etch" the cuvette windows and mess up your readings.
13. Read the absorbance at 420nm. These values reflect the amount of yellow color in each tube.
14. Read the absorbance of each at 550 nm. These values reflect the amount of cell debris and differences in the plastic cuvettes themselves.
15. Dispose of your samples properly: liquid contents of cuvettes can go down the sink, empty cuvettes can go in the sharps containers, eppendorfs with CHCl₃ can go into a waste bottle in the hood.
16. The β-gal activity in each sample is reported as "Miller Units" according to the following formula:

where:
Abs 420 is a measure of the yellow color produced by the β-gal activity.
Abs 550 is a measure of cell debris.
OD 600 is a measure of the cell density.
t is the reaction time from start to stop, measured in minutes.
v is the culture volume that you added to the reactions, measured in mls.
You can average the duplicate values for each sample unless you know of a reason not to (e.g. one tube spilled or had the incorrect amount of something added to it...)

Part 3: Observe solid cultures

For next time