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Dual Luciferase Assay Protocol (Promega E1910)

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Inoculate 3-5 ml of medium that will be used for assay (except for CR experiments) with pVW31 transformants. Shake at 30 degrees C overnight. The medium will usually be MinD+    but could be YEP, C-URA, etc.

The next morning, use the Nanodrop to get the A600.  For instructions on how to do this, see Measuring OD600 on the Nanodrop.  Dilute cells to A600 = 0.2/ml in 4 ml of, typically, MinD+.  This means your  starting OD/ml is (whatever the Nanodrop said) per 1ml (because the cuvette is 1 ml).

EXAMPLE CALCULATION:  You get a Nanodrop OD600 aka A600 reading of 0.4.  This is 0.4/ml since you read 1ml.  You need 0.2/ml * 4ml, or 0.8 OD600 worth of yeast, so spin down 2mls of your overnight (2mls * your overnight OD of 0.4/ml = 0.8), then resuspend this pellet in 4ml of MinD+ to have 0.2/ml yeast in your MinD+.  We have a spreadsheet with formulas typed in for this as well. REMEMBER: this example calculation works if your yeast right out of the tube is in the 0.1-1 range for OD600.   If not, you will have to dilute the yeast, get diluted OD600, calculate undiluted OD600, and go from there, as detailed under Measuring OD600 on the Nanodrop.

Now, incubate with shaking for 4 hrs at 30C.

After 4 hours, check A600 again, and spin down a total of 0.22 A600 in microfuge, at top speed for 2 min at room temp.

Again, EXAMPLE CALCULATION: You check your MinD+ culture after shaking in the Nanodrop, and get 0.3 in 1ml cuvette, or 0.3/ml.  REALITY CHECK: you just put in 0.2/ml 4 hours ago, so if you read <0.2ml after 4 hours of shaking you're doing something wrong!  Okay.  So you have 0.3/ml- you want a total of 0.22, so you spin down 0.22*1ml/0.33 = 2/3 ml or 667ul to get the pellet.

(A600 from 4 hour cultures) * (XmL) = (0.22) * (1mL)

Carefully remove all medium from this spin down, and resuspend the cells in 500 ul of 1X Promega Passive Lysis Buffer at room temp. (Note: the 5X PLB or Passive Lysis Buffer is stored at -20C but is not frozen; dilute to 1X with water.  we have extra backup PLB in the -80 as well.) Let cells lyse for 30 min at room temp.

In the meantime, thaw out the other reagents for the dual luciferase assay:

The dual luciferase assay uses two solutions: the Luciferase Assay solution (1) and the Stop n' Glo solution (2).  Both of these solutions are provided as two separate parts in the Promega kit.  You have to mix them both up when you open a new kit. For solution 1, resuspend all of the Luciferase Assay Substrate in the full 10 ml of Luciferase Assay Buffer II and store in the screw-cap white plastic vial the Luciferase Assay Buffer II came in. Store leftovers at -80C.

For solution 2, resuspend all of the Stop n' Glo substrate in the Stop n' Glo buffer, and store in the screw-cap white plastic vial the Stop n' Glo buffer came in. Store leftovers at -80C.

After the 30 min lysis step, pipet 5 ul of lysed cell samples into wells of black 96-well plates (Nunc 267342 U96 PP 0.5ml). All assays are done in triplicate. With the pVW31 setup (GCN4 promoter and 5' driving the Gcn4-firefly luciferase expression and the strong PGK1 promoter driving the Renilla luciferase expression), the Renilla levels are much higher and, because of light scattering, can interfere with the much lower firefly reading of the next sample. Therefore, you need to space the samples out in the 96-well format, as shown in the figure following. Use the Dual Luc protocol on our Victor X3 luminometer; additionally, a luminometer templatemay be easily downloaded in .ods format.

Figure: layout for 8 samples in triplicate in 1 96-well plate, with 2 PLB-only (5ul of PLB) control wells.

1

2

3

4

5

6

7

8

9

10

11

12

A

PLB

1

1

1

2

2

2

B

C

3

3

3

4

4

4

D

E

PLB

F

5

5

5

6

6

6

G

H

7

7

7

8

8

8

These reagents plus the Passive Lysis Buffer are from the Promega Dual-Luciferase Reporter Assay Kit (Product number E1910, E1960 and E1980). The Promega Technical Abstract / Protocol is here, which links to the kit product pages.

The protocol you want to run on our Victor X3 with dual injectors is normally titled "DLR". You should check that the protocol has not been edited before running it; the icon for the PerkinElmer 2030 manager ids on the desktop, then select the DLR protocol and click the little lab notebook icon.

It should read:

DISPENSE Injector = 1, Speed 5, Volume = 40ul, Increment 0ul, Replicate =1, Injection mode = aspVol=dispVol, Repeated operation = Yes

SHAKE Shaking duration = 1s, Shaking speed = Fast, Shaking diameter = 1.00mm, Shaking type = Linear, Repeated operation = Yes

LUM 1S

DISPENSE Injector = 2, Speed 5, Volume = 40ul, Increment 0ul, Replicate =1, Injection mode = aspVol=dispVol, Repeated operation = Yes

SHAKE Shaking duration = 1s, Shaking speed = Fast, Shaking diameter = 1.00mm, Shaking type = Linear, Repeated operation = Yes

LUM 1S.

To run, you will need to make the Luciferase Assay solution the source for Injector 1, and the Stop n' Glo solution the source for Injector 2.

When the run has finished, save latest results as an .xls file and put it in the Dropbox immediately, and email a copy to yourself and anyone working on the experiment with you. Filenames must always contain the date and your initials, and if you are running multiple experiments or plates in one day this must be clear in each file name.

WHEN YOU ARE DONE ALWAYS CLEAN THE INJECTORS. Clean by running Fill, then Empty, then Flush from the injector control button on the software, first with 70% EtOh and then with Baxter water. Use a separate tube (15ml conical is fine) for each injector as you never want any traces of Stop n' Glo from injector 2 to get into injector 1.