text stringlengths 0 4.18k |
|---|
Tab 5 Text |
Tab 6 Text |
## 3.3 GEX Adaptor Ligation |
- a. Prepare Adaptor Ligation Mix. Pipette mix and centrifuge briefly. |
- b. Add 50 μl Adaptor Ligation Mix to 50 μl sample. Pipette mix 15x (pipette set to 90 μl). Centrifuge briefly. |
- c. Incubate in a thermal cycler with the following protocol. |
<!-- image --> |
| Adaptor Ligation Mix Add reagents in the order listed | PN | 1X ( μ l) | 4X + 10% ( μ l) | 8X + 10% ( μ l) | |
|---------------------------------------------------------|---------------|-------------|-------------------|-------------------| |
| Ligation Mix | 2001109 | 40 | 176 | 352 | |
| DNA Ligase | 220110/220131 | 10 | 44 | 88 | |
| Total | | 50 | 220 | 440 | |
| Lid Temperature | Reaction Volume | Run Time | |
|--------------------------------------------------|-------------------|---------------| |
| 30°C off if the instrument does not enable 30°C) | 100 μ l | 15 min | |
| Step | Temperature | Time hh:mm:ss | |
| 1 | 20°C | 00:15:00 | |
| 2 | 4°C | Hold | |
Tab 1 Text |
Tab 2 Text |
Tab 3 Text |
Tab 4 Text |
Tab 5 Text |
Tab 6 Text |
## 3.4 GEX Post Ligation Cleanup - SPRIselect |
- a. Vortex to resuspend SPRIselect Reagent. Add 80 μl SPRIselect Reagent (0.8X) to each sample. 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. 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 30.5 μl Buffer EB. Pipette mix 15x. |
- k. Incubate 2 min at room temperature . |
- l. Place on the magnet· Low until the solution clears. |
- m. Transfer 30 μl sample to a new tube strip. |
Tab 1 Text |
Tab 2 Text |
Tab 3 Text |
Tab 4 Text |
Tab 5 Text |
Tab 6 Text |
## 3.5 GEX Sample Index PCR |
<!-- image --> |
<!-- image --> |
<!-- image --> |
- a. Choose the appropriate sample index sets to ensure that no sample indices overlap in a multiplexed sequencing run. Record the 10x sample index name (PN-3000431 Dual Index Plate TT Set A well ID) used. |
- b. Add 50 μl Library Amp Mix (PN-2000531) or Amp Mix (PN2000047/2000103) to 30 μl sample. |
- c. Add 20 μl of an individual Dual Index TT Set A to each sample and record the well ID used. Pipette mix 5x (pipette set to 90 μl). Centrifuge briefly. |
- d. Incubate in a thermal cycler with the following protocol. |
| Lid Temperature | Reaction Volume | Run Time | |
|-------------------|-----------------------------------------|---------------| |
| 105°C | 100 μ l | ~30 min | |
| Step | Temperature | Time hh:mm:ss | |
| 1 | 98°C | 00:00:45 | |
| 2 | 98°C | 00:00:20 | |
| 3 | 54°C | 00:00:30 | |
| 4 | 72°C | 00:00:20 | |
| 5 | Go to step 2, see below for # of cycles | | |
| 6 | 72°C | 00:01:00 | |
| 7 | 4°C | Hold | |
The table recommends a starting point for optimization. The total cycles should be optimized based on 25% carry forward cDNA yield/input calculated during cDNA QC & Quantification (step 2.4). |
## Recommended Cycle Numbers |
| cDNA Input | Total Cycles | |
|----------------|----------------| |
| 0.25-50 ng | 14-16 | |
| 50-250 ng | 12-14 | |
| 250-600 ng | 10-12 | |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.