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| 600-1,100 ng | 8-10 | |
| 1,100-1,500 ng | 6-8 | |
| >1,500 ng | 5 | |
- e. Store at 4°C for up to 72 h or proceed to the next step. |
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## 3.6 Post Sample Index PCR Double Sided Size Selection SPRIselect |
- a. Vortex to resuspend the SPRIselect reagent. Add 60 μl SPRIselect Reagent (0.6X) to each sample. Pipette mix 15x (pipette set to 150 μl). |
- b. Incubate 5 min at room temperature . |
- c. Place the magnet· High until the solution clears. DO NOT discard supernatant. |
- d. Transfer 150 μl supernatant to a new tube strip. |
- e. Vortex to resuspend the SPRIselect reagent. Add 20 μl SPRIselect Reagent (0.8X) to each transferred supernatant. Pipette mix 15x (pipette set to 150 μl). |
- f. Incubate 5 min at room temperature . |
- g. Place the magnet· High until the solution clears. |
- h. Remove 165 μl supernatant. DO NOT discard any beads. |
- i. With the tube still in the magnet, add 200 μl 80% ethanol to the pellet. Wait 30 sec . |
- j. Remove the ethanol. |
- k. Repeat steps i and j for a total of 2 washes. |
- l. Centrifuge briefly. Place on the magnet· Low . |
- m. Remove remaining ethanol. DO NOT over dry beads to ensure maximum elution efficiency. |
- n. Remove from the magnet. Add 35.5 μl Buffer EB. Pipette mix 15x (pipette set to 35 µ l). |
- o. Incubate 2 min at room temperature . |
- p. Place on the magnet· Low until the solution clears. |
- q. Transfer 35 μl to a new tube strip. |
- r. Store at 4°C for up to 72 h or at -20°C for long-term storage. |
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## 3.7 Post Library Construction QC |
## Library QC |
Use Agilent Bioanalyzer, Perkin Elmer LabChip, or Agilent Tapestation for QC. |
- a. Run 1 μl sample at 1:10 dilution on an Agilent Bioanalyzer High Sensitivity chip. |
- a. Select the region between 200-2,000 bp to determine average size of the library. This will be used as the insert size for library quantification. |
## Representative Trace |
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- If additional peaks below 200 bp are observed, repeat step 3.6 Post Sample Index PCR Double Sided Size Selection - SPRIselect. Add nuclease-free water to bring the library volume to 100 μl before performing step 3.6a. Note that ~40% of material may be lost when repeating step 3.6. Alternatively, libraries that will ... |
See Appendix on page 72 for representative traces |
## Quantification |
Library quantification should be performed prior to sequencing. For the most accurate quantitative assessment of libraries, a qPCR-based method should be used to ensure that the sequencing flowcell is loaded properly. |
See Post Library Construction Quantification using KAPA qPCR on page 74 |
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## Step 4: |
## Sequencing |
| Sequencing Libraries | 62 | |
|-------------------------------------------|------| |
| Illumina Sequencer Compatibility | 62 | |
| Sample Indices | 62 | |
| Library Sequencing Depth & Run Parameters | 63 | |
| Library Loading | 63 | |
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