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| 7 | 4°C | Hold | |
Recommended starting point for cycle number optimization. The optimal cycle number is a trade-off between generating sufficient final mass for libraries & minimizing PCR amplification artifacts. |
| Targeted Cell Recovery | Total Cycles | |
|--------------------------|----------------| |
| <500 | 13 | |
| 501-6,000 | 12 | |
| >6,000 | 11 | |
- e. Store at 4°C for up to 72 h or -20°C for ≤1 week , or proceed to the next step. |
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## 2.3 cDNA Cleanup - SPRIselect |
- a. Vortex to resuspend the SPRIselect reagent. Add 60 μl SPRIselect reagent (0.6X) to each sample and pipette mix 15x (pipette set to 150 μl). |
- b. Incubate 5 min at room temperature . |
- c. Place on the magnet· High until the solution clears. |
- d. Remove the supernatant. |
- e. Add 200 μl 80% ethanol to the pellet. Wait 30 sec . |
- f. Remove the ethanol. |
- g. Repeat steps e and f for a total of 2 washes. |
- h. Centrifuge briefly and place on the magnet· Low . |
- i. Remove any remaining ethanol. Air dry for 2 min . DO NOT exceed 2 min as this will decrease elution efficiency. |
- j. Remove from the magnet. Add 40.5 μl Buffer EB. Pipette mix 15x. |
- k. Incubate 2 min at room temperature. |
- l. Place the tube strip on the magnet· High until the solution clears. |
- m. Transfer 40 μl sample to a new tube strip. |
- n. Store at 4°C for up to 72 h or -20°C for ≤4 weeks , or proceed to the next step. |
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## 2.4 Post cDNA Amplification QC & Quantification |
- a. Run 1 μl (see dilutions below) product from step 2.3 on an Agilent Bioanalyzer High Sensitivity chip. |
2. l Low RNA content cells (<1 pg total RNA/cell) should be run undiluted |
3. l High RNA content cells should be run at 1:5 or 1:10 dilution |
- b. See example calculation in the following page. |
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## Alternate Quantification Methods |
Agilent TapeStation |
LabChip |
See Appendix on page 72 for representative traces. |
## Example Calculation |
- i. Select Region: Under the 'Electropherogram' view, choose the 'Region Table.' Manually select the region of ~200 - ~9000 bp. |
- ii. Note Concentration [pg/μl] |
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- iii. Calculate: Multiply the diluted sample concentration [pg/μl] reported via Agilent 2100 Expert Software by the dilution factor and divide by 1000 to obtain the total cDNA yield in ng/μl. |
Carry forward ONLY 25% of total cDNA yield into Gene Expression Library Construction. |
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