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Use a thermal cycler that can accommodate at least 100 μl volume. A volume corresponding to the maximum allowable amount, for a given supported thermal cycler, is the preferred setting. For example a volume of 125 μl is the preferred setting on Bio-Rad C1000 Touch. In alternate thermal cyclers, use highest reaction v... |
- a. Incubate in a thermal cycler with the following protocol. |
b. Store at 4°C for up to 72 h or at -20°C for up to a week , or proceed to the next step. |
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## Step 2: |
## Post GEM-RT Cleanup & cDNA Amplification |
| 2.0 Get Started | 42 | |
|-------------------------------------|------| |
| 2.1 Post GEM-RT Cleanup - Dynabeads | 43 | |
| 2.2 cDNA Amplification | 46 | |
| 2.3 cDNA Cleanup - SPRIselect | 47 | |
2 |
2.4 Post cDNA Amplification QC & Quantification |
48 |
## 2.0 Get Started |
| Item | | 10x PN Preparation & Handling | Storage ... |
|---------------------------------|-----------------------------------------------------------------------------------------------------------------------------------|---------------------------------|-----------------------------------------------------------------------------------------------------------------------... |
| Equilibrate to Room Temperature | Equilibrate to Room Temperature | Equilibrate to Room Temperature | Equilibrate to Room Temperature ... |
| □ | Reducing Agent B | 2000087 | Thaw, vortex, verify no precipitate, centrifuge briefly. ... |
| □ | cDNA Primers | 2000089 | Thaw, vortex, centrifuge briefly. ... |
| 2000089 2000439/ □ | Dynabeads MyOne SILANE | 2000048 | Vortex thoroughly (≥30 sec) immediately before adding to the mix. If still clumpy, pipette mix to resuspend completely... |
| □ | Beckman Coulter SPRIselect Reagent | - | Manufacturer's recommendations. ... |
| □ | Agilent Bioanalyzer High Sensitivity Kit If used for QC & quantification | - | Manufacturer's recommendations. ... |
| □ | DNA High Sensitivity Reagent Kit If LabChip used for QC & quantification | - | Manufacturer's recommendations. ... |
| □ | Agilent TapeStation ScreenTape & Reagents If used for QC & quantification | - | Manufacturer's recommendations. ... |
| Place on Ice | Place on Ice | Place on Ice | Place on Ice ... |
| □ | Amp Mix DO NOT use the Library Amp Mix PN-2000531 (if provided) at this step to avoid a significant decrease in assay performance | 2000047 /2000103 | Vortex, centrifuge briefly. ... |
| Thaw at 65°C | Thaw at 65°C | Thaw at 65°C | Thaw at 65°C ... |
| □ | Cleanup Buffer | 2000088 | Thaw for 10 min at 65°C at max speed on a thermomixer. Verify no visible crystals. Cool to room temperature. ... |
| Obtain | Obtain | Obtain | Obtain ... |
| □ | Recovery Agent | 220016 | - ... |
| □ | 10x Magnetic Separator B | 2001212 | - ... |
| □ | Qiagen Buffer EB | - | Manufacturer's recommendations. ... |
| □ | Bio-Rad 10% Tween 20 | - | Manufacturer's recommendations. ... |
| □ | Prepare 80% Ethanol Prepare 15 ml for 8 reactions. | - | Prepare fresh. ... |
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## 2.1 Post GEM-RT Cleanup - Dynabeads |
- a. Add 125 μl Recovery Agent to each sample (Post GEM-RT) at room temperature. If using sample stored at -20°C, equilibrate to room temperature before adding the Recovery Agent . DO NOT pipette mix or vortex the biphasic mixture. Wait 2 min . |
The resulting biphasic mixture contains Recovery Agent/Partitioning Oil (pink) and aqueous phase (clear), with no persisting emulsion (opaque). |
## If biphasic separation is incomplete: |
- l Firmly secure the cap on the tube strip, ensuring that no liquid is trapped between the cap and the tube rim. |
- l Mix by inverting the capped tube strip 5x, centrifuge briefly, and proceed to step b. DO NOT invert without firmly securing the caps. |
A smaller aqueous phase volume indicates a clog during GEM generation. |
- b. Slowly remove and discard 125 μl Recovery Agent/Partitioning Oil (pink) from the bottom of the tube. DO NOT aspirate any aqueous sample. |
- c. Prepare Dynabeads Cleanup Mix. |
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Before using Dynabeads MyOne SILANE to prepare the Dynabeads Cleanup Mix: |
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l |
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