Amplify SNT

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Consumables

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4X PCR Master Mix (PMM4)

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Washing Buffer (PWB1)

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WTA-F-V2

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One of the following additive primer options:

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Additive Primer A

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Additive Primer H

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Additive Primer A and H

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0.2 ml 8-tube strips and caps

Preparation

PMM4—Pipette to mix, and then centrifuge briefly.

Save the following SNT Amplification program on the thermal cycler:

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Set the lid temperature to 105°C

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95°C for 3 minutes

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10–14 cycles of:

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98°C for 15 seconds

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69°C for 1 minute 20 seconds

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72°C for 5 minutes

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Hold at 4°C

Adjust the program cycle number to optimize for sample input.

Annealing and extension both occur at 69°C in this profile.

Procedure

Add SNT amplification reagents directly to each sample using one of the following tables:

SNT Amplification Master Mix for Additive Primer A or Additive Primer H
Reagent	Volume Per Reaction (μl)
PMM4	375
WTA-F-V2 at 20 µM	37.5
Diluted Additive Primer A or H or similar at 10 µM	37.5
0.5X PWB1	50
Total	500

SNT Amplification Master Mix for Additive Primer A and Additive Primer H
Reagent	Volume Per Reaction (μl)
PMM4	375
WTA-F-V2 at 20 µM	37.5
Diluted Additive Primer A or similar at 10 µM	37.5
Diluted Additive Primer H or similar at 10 µM	37.5
0.5X PWB1	12.5
Total	500

Flick to mix, and then pulse vortex for 5 seconds.

Pipette 10 times to resuspend.

Distribute the SNT reaction mixture into 8 x 188 μl aliquots in an 8-tube strip. Centrifuge the SNT reaction mixture briefly before dispensing the last aliquot, and then transfer all the remaining volume.

Using a 200 µl multichannel pipette, transfer 62 μl SNT reaction mixture from each aliquot into two new 8-tube strips, for a total of three 8-tube strips per sample.

Use the same tips between transfers to avoid loss of PIPs.

Label the 8-tube strips appropriately so each sample can be recombined after SNT amplification.

Place on the preprogrammed thermal cycler and run the SNT Amplification program.

SAFE STOPPING POINT

If you are stopping, store samples overnight at 2°C to 8°C.