The DLR™ assay from Promega is a commonly used assay to measure gene transcription and control and is one of the most commonly used luminescence-based assays in microplates. This protocol describes the use of BioTek's Synergy™ 2 with injectors for the DLR assay.
Follow the steps below to create a DLR protocol in BioTek's Gen5™ software (Figure 1).
In Gen5, select File/New Protocol.
Open the Procedure in the Protocol tree on the left of the screen.
Click on the Well button to create a well mode block: the reader will process the sequence on each well before moving to the next well.
Click on Dispense to program the first dispense step (LAR II re-agent). Select dispenser #1, adjust dispense volume as required, and select the portion of the plate to be processed using the Full Plate button. All other parameters can be left at their default value
* *The Help button available on most Gen5 screens provides context sensitive information, should you need more details about the different parameters available.
Click Delay and adjust the time to 2 seconds from the end of previous step (default selection). Click OK.
Click Read and adjust the parameters as follows: detection method is luminescence, integration time is 10 seconds (typically), emission should say Hole, sensitivity should be set to 150 (see sensitivity note below). All other parameters can be left at their default value
* . Click OK.Repeat step 4, using dispenser 2 for the Stop and Glo reagent.
Repeat step 5 to add a 2-second delay after the second injection.
Click Read: a limited form is displayed, giving access to the step label and Integration time. Just validate with OK.
In the Procedure screen, click OK to validate.
Data Analysis:
Open the Data Reduction in the Protocol tree.
Click on Transformation then Select Multiple Data Sets.
Select the DS2 radio button. Set DS1 to Read1 and DS2 to Read2.
New Data Set Name is DLR Ratio.
Check Use single formula for all wells.
Current Formula is DS1/DS2.
Click OK.
Save the Gen5 protocol (File/Save).
Sensitivity parameter: gain applied to the detector. A value of 150 accommodates most luminescence assays. To adjust this parameter, run the protocol on two high wells (high signal expected). If the result is below 100,000 RLU, increase the sensitivity and try again. If the result is above 2,500,000 RLU (or overflow), decrease the sensitivity and try again.
Dual-Luciferase is a trademark of Promega Corporation and is registered with the U.S. Patent and Trademark office.
*The Help button available on most Gen5 screens provides context sensitive information, should you need more details about the different parameters available.
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