SYBR^® Green dye detection protocol
for standard or mid-throughput

1. After PCR, add 1/10 volume of 100X SYBR^® Green I Nucleic Acid Gel Stain for a 10X final concentration. (Stock is 10,000X, Life Technologies, Cat. # S-7563). Be careful not to cross contaminate samples; after sealing the plate, briefly centrifuge at 1200 x g, vortex, and centrifuge again.
2. Incubate the plate in a thermocycler at 72°C for 1 min to denature primer dimers, then immediately visualize using a blue-light transilluminator and yellow/orange filter or see SYBR^® Green manual for recommendations.
3. Take a picture to document results. A SYBR compatible filter on the camera is necessary if not already in use for visualization. Below is an example of Positive Controls and Negative or No Template Control (NTC) in a 96 well plate format.


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4. Alternatively, a plate spectrophotometer with fluorescence capabilities can be used to capture signal intensities after excitation and emission using instrument-dependent SYBR^® green wavelengths (excitation λ max = 497 nm, emission λ max = 520 nm).
5. Confirm atypical genotypes or questionable signal intensities by gel electrophoresis. Add 1/10 volume of loading buffer and load 20 μL of sample onto a 1% agarose 1X TAE gel. SYBR^® Safe stain is not needed in the gel, but 1/10 volume of SYBR® Green should be added to the molecular weight marker.
6. Visualize and document as in steps 2 and 3.

Standard Agarose Gel Protocol  |  Mid-throughput Protocol

Mid-throughput Protocol
(setting up 1 or 4, 96 well plates)

Setting up PCR Reaction

1. Dilute Sample DNA (S1, S2, S3 …) to 5 ng/μL in a 12 μL volume in the orientation shown below. Also add 12 μL of controls D, MP, AP and Neg. (Negative control, template/nuclease-free water) to designated wells.

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2. Make 4 primer master mixes by combining the volumes shown below. Use multiples or combinations of volumes listed for more than 4 plates.

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3. Arrange primer mixes in a tube rack, then use a multichannel pipette with 4 alternating tips to pipette 57.5 μL (single plate PCR set up) or 235 μL (4 plate PCR set up) to 4 columns of a PCR plate (as shown at the top of the following page). Alternatively a template/nuclease-free reagent reservoir can be used for each primer mix when running multiple PCR plates. Volumes can be adjusted for number of plates and best use of overages.

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4. Use a multichannel repeat pipette to draw up 10.5 μL of DNA from column 1 of the DNA plate (from step 1), discard 2 μL back into wells, and then dispense 2 μL to columns 1-4 of PCR plate 1. Similarly, complete the rest of PCR Plate 1, and Plates 2, 3, and 4 according to diagram below.

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5. Use a multichannel manual pipette to transfer 18 μL Primer 1 Mix from Column 2 of staging plate to columns 1, 5 and 9 on PCR plates 1 through 4. Similarly add Primer mixes 2, 3 and 4 according to the diagram below. After each addition mix by pipetting up and down 3 times, and discard tips. Do not blow out sub-microliter volume remaining in the end of the tips, as this could cross contaminate sample wells. Label edge of plates with 1’s, 2’s, 3’s and 4’s to avoid confusion during pipetting.

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6. Seal plate , centrifuge briefly at 1200 x g and run PCR with the program below.

• Step 1:  94°C  2 min
• Step 2:  94°C  30 sec
• Step 3:  58°C  1 min
• Step 4:  72°C  1 min
• Step 5:  Go to step 2 for 34 cycles
• Step 6:  72°C  5 min
• Step 7:  4°C  on hold

7. After PCR, add 1/10 volume of 10X loading buffer before loading on an agarose gel.

8. Load 20 μl PCR product onto a 1X TAE 1% agarose gel containing 1X SYBR Safe DNA Gel Stain (Life Technologies, Cat. # S33102).

9. Electrophorese for 60 min at 110V.

10. Visualize PCR bands using a blue-light transilluminator and yellow/orange filter or see SYBR safe manual for recommendations.

Standard Agarose Gel Protocol  |  SYBR^® Green dye detection protocol