Picogreen on the Real TIme PCR Machine | Ambrosia Symbiosis

Picogreen on the RT PCR Machine

Per Chris Dervinis, Sr Biologist and Forest Genomics Lab Manager, SFRC, Univ. of FL :

“We use the sybr green filters for the assay, which is not mentioned in the protocol. One thing to keep in mind is that the linear range for picogreen in these smaller assay is lower than it is with a plate reader and larger volume size, so you will need to be sure that your samples are under 50ng/ul or you will need to dilute for an accurate approximation.”

Plate Setup

1.Set up the standards using the Lambda DNA – Component C (100 ng/µl) with a total volume of 5 µl

For example: Make a 10 ng/µl Standard from the Lambda DNA

Add TE to the plate, according to the scheme below:

0 µl

4 µl

1 µl

2 µl

2.5 µl

3 µl

3.5 µl

4 µl

5 µl

Add DNA Standard (10 ng/ μL – kit supplies DNA standard 100 ng/ μL) and the samples, according to the scheme bellow:

5 µl (50ng)

5 µl

1 μL

4 µl (40 ng)

4 µl

3 µl (30 ng)

3 µl

2.5 µl (25 ng)

2.5 µl

2 µl (20 ng)

2 µl

1.5 µl (15 ng)

1.5 µl

1 µl (10 ng)

1 µl

0 µl (0 ng)

0 µl

1.Add 25 µl of Picogreen solution into each well

Picogreen dilution: (1 Component A: 200 TE)

NOTE: Protect Picogreen solution from light (photo-sensitive)

1.Add the PCR flat caps over each column with the Strip Cap Tool (avoid touching the top of caps with your hands because the reading must pass through the caps)

Vortex and spin down

RT PCR Set up

Lamp Warm up takes 20 mins so have the lamp warming up before starting plate setup.

1.Open the realplex program (password is “e”). If the program is not recognizing the thermocycler, make sure the USB connection to the computer is in the right port (both the port and the cord are marked with marker). All other connections can be checked based on the arrangement in the manual (on computer desktop).

Choose “Quantitative Plate Read”
Set the plate grid
1. Highlight wells that are for standard and click on “Standard” (right hand side)
2. Highlight wells that are for samples and click on “Unknown” (right hand side)
3. Highlight ALL wells that will be read and click SYBR
4. Set the values for the Standards (for example: 50, 40, 30, 25, 20, 15, 10, 0)
  1. Set the 2 standard as replicates (right hand side) so the 2 readings will be averaged for 1 point on the standard curve
  2. Change the quantity from “copies” to “nanogram” (right hand side)
    1. It will ask for the conversion; Click “Do Not Convert”
    2. Click Run
    3. Click Pre-Read
    4. To analyze the data, click “Analysis” on the top right hand side, click “Results” tab to view Standard curve, Text Report, etc.

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