PCR and Index Addition Pool 1, 1.5X clean pools 1,3,4,5, qubit, gel
PCR for Pool 1, 1.5X clean, qubit, gel pools 1,3,4,5
Date Performed: February 1st, 2022
PCR and Index Addition Pool 1
Notes on PCR
Pool 1 Index Pair = 502/702
1.5X Bead Clean on Pools 1,3,4,5
- Take out KAPA Pure Beads ~30 minutes before use and make fresh 80% ethanol
- Combine the 6 reactions into a single 1.5 mL tube, for a total of 120μl
- Add 1.5X of KAPA Pure Beads (180μl) to each pooled tube, mix by pipetting, and incubate on a shaker for 15 minutes
- Put the tubes on the long magnet rack, wait until the liquid becomes clear, and remove as much of the clear liquid as you can
- Add 1000μl 80% EtOH to each tube while keeping it on the magnet
- Remove 1000μl of the EtOH
- Add 1000μl 80% EtOH to each tube
- Remove ALL EtOH carefully from each tube, use the p20 or p200 to remove any excess ethanol
- Let samples sit for ~5 minutes, letting the ethanol evaporate, but the beads not dry out
- Take tubes of the magnet stand and elute the DNA in 35μl 10mM Tris HCl and incubate shaking for 5 minutes
- Place the tubes back in the magnet, and wait until the liquid goes clear. Remove all liquid and put into new strip tubes: this is your library!
2X Qubit for each pool
Sample | Run 1 | Run 2 |
---|---|---|
S1 | 440.54 | |
S2 | 107362.69 | |
Pool 1 | 4.94 | 4.90 |
Pool 3 | 7.32 | 7.26 |
Pool 4 | 7.72 | 7.78 |
Pool 5 | 10.9 | 10.8 |
Sample | Run 1 | Run 2 |
---|---|---|
S1 | 413.73 | |
S2 | 108102.91 | |
Pool 1 | 5.24 | 5.24 |
Pool 3 | 7.40 | 7.38 |
Pool 4 | 7.98 | 7.80 |
Pool 5 | 10.6 | 10.7 |
12 cycle PCR on 1ul from size selected pools 1,3,4,5 for gel
Each PCR Tube for Pools 1,3,4,5
* 1 ul sample
* 10ul Kapa Hifi
* .5 ul 505 primer
* .5 ul 705 primer
* 8ul nuclease free water
Pool 1 = 502/702 Pool 3 = 506/706 Pool 4 = 507/707 Pool 5 = 508/708
Written on February 1, 2022