Page Comparison

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50 uM PNA blockers mPNA and pPNA

12 6 uM allows for 2. 5 ul per combined PNA to reach 2 uM and 01.625 25 ul for 0.5 uM

24 12 ul each PNA in 76 ul TE makes 100 ul 12uM 6uM PNAs with both m and p

Dilute DNAs to 10 ng/ul

Use concentrations provided

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• Add 6 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed label “NS5 PCR”. ( one needs 2 ul of template and 2 ul of the primers).

PNA volumes

Water volumes

PCR

Run STD on Thermocycler Program GSAF36:

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• Equilibrate Beads to room Temperature

• Add 24 ul of MagBeads to each well and 15 ul of replicate to same well of replicate

• Pipette mix up and down 10 times.

• Incubate at RT for 5 minutes

• Secure plate on magnet plate; incubate at RT for 5 minutes (until wells are clear)

• Remove 65 ul from each well; keep tips to left or right depending on the column to avoid bead pellet.

• Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well

• Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well

• Reaspirate from each well to assure maximum EtOH removal

• Allow plate to air dry for 7 minutes.

• Remove sample plate from magnet plate.

• Add 40 ul TE; pipette mix 10+ times. Incubate 2 minutes at RT.

• Place sample plate back on magnet for 5 minutes or until all wells are cleared.

• Transfer 40 ul to labeled transparent plate (Plate name_PCR_MIDs)

• Store in -20 until ready to qPCR

TapeStation

Pool:

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Samples:

Pool, followed by Brock 2, column 2 (sample 5 is the blank)

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Blank:

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qPCR

45 nM

2.2 ul sample plus 97.8 ul RSB to make 1 nM

Sequence Dilution

Dilute to 1 nM:

2.2 ul sample plus 97.8 ul RSB

Add 10% PhiX and dilute to 65 pM:

13 ul sample plus 1.3 ul PhiX plus 185.7 RSB