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Vortex the Dynabeads thoroughly ( ≥30 sec ) immediately before adding to the mix. |
- l Aspirate full liquid volume in the Dynabead tube with a pipette tip to verify that beads have not settled in the bottom of the tube. If clumps are present, pipette mix to resuspend completely. DO NOT centrifuge before adding to the mix. |
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- d. Vortex and add 200 μl to each tube. Pipette mix 10x (pipette set to 200 μl). Before adding ensure that there are no clumps. |
- e. Incubate 10 min at room temperature (keep caps open). Pipette mix again at ~5 min after start of incubation to resuspend settled beads. |
- f. Prepare Elution Solution I. Vortex and centrifuge briefly. |
- g. At the end of 10 min incubation, place on a 10x Magnetic Separator· High position (magnet· High ) until the solution clears. |
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A white interface between the aqueous phase and Recovery Agent is normal. |
- h. Remove the supernatant. |
- i. Add 300 μl 80% ethanol to the pellet while on the magnet. Wait 30 sec . |
- j. Remove the ethanol. |
- k. Add 200 μl 80% ethanol to pellet. Wait 30 sec . |
- l. Remove the ethanol. |
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- m. Centrifuge briefly. Place on the 10x Magnetic Separator· Low position (magnet· Low) . |
- n. Remove remaining ethanol. Air dry for 1 min . |
- o. Remove from the magnet. Immediately add 35.5 μl Elution Solution I. |
- p. Pipette mix (pipette set to 30 μl) without introducing bubbles. Pipette mix 15x. If beads still appear clumpy, continue pipette mixing until fully resuspended. |
- q. Incubate 1 min at room temperature . |
- r. Place on the magnet· Low until the solution clears. |
- s. Transfer 35 μl sample to a new tube strip. |
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## 2.2 cDNA Amplification |
- a. Prepare cDNA Amplification Mix on ice. Vortex and centrifuge briefly. |
- b. Add 65 μl cDNA Amplification Reaction Mix to 35 μl sample (Post-GEM-RT Cleanup, step 2.1s). |
- c. Pipette mix 15x (pipette set to 90 μl). Centrifuge briefly. |
- d. Incubate in a thermal cycler with the following protocol. |
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| cDNA Amplification Reaction Mix Add reagents in the order listed | cDNA Amplification Reaction Mix Add reagents in the order listed | PN | 1X ( μ l) | 4X + 10% ( μ l) | 8X + 10% ( μ l) | |
|--------------------------------------------------------------------|--------------------------------------------------------------------|------------------|-------------|-------------------|-------------------| |
| | Amp Mix DO NOT use the Library Amp Mix PN-2000531 (if provided) | 2000047/ 2000103 | 50 | 220 | 440 | |
| | cDNA Primers | 2000089 | 15 | 66 | 132 | |
| | Total | | 65 | 286 | 572 | |
| Lid Temperature | Reaction Volume | Run Time | |
|-------------------|-----------------------------------------------------|-----------------------------------------------------| |
| 105°C | 100 μ l | ~25-50 min | |
| Step | Temperature | Time hh:mm:ss | |
| 1 | 98°C | 00:00:45 | |
| 2 | 98°C | 00:00:20 | |
| 3 | 63°C | 00:00:30 | |
| 4 | 72°C | 00:01:00 | |
| 5 | Go to Step 2, see table below for total # of cycles | Go to Step 2, see table below for total # of cycles | |
| 6 | 72°C | 00:01:00 | |
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