Additional 4 cycles PCR for all pools
Ran 4 more cycles PCR for all pools (1-5) due to low DNA concentration
Date Performed: February 24th, 2022
Second Round PCR
- Made 4 strip tubes each for 5 pools
- Added 13ul master mix to each tube/pool
- Added 7ul sample to each tube/pool
- Ran for 4 cycles PCR
Primer Pairs
- Pool 1 502/702
- Pool 2 505/705
- Pool 3 506/706
- Pool 4 507/707
- Pool 5 508/708
Master Mix (One Per Pool)
- 10ul kapa hifi x 4 x 1.1 = 44ul Kapa Hifi
- 2ul nuclease free water x4 x 1.1 = 8.8ul nuclease free water
- .5ul 50X primer x 4 x 1.1 = 2.2ul
- .5ul 70X primer x 4 x 1.1 = 2.2ul
Post PCR 1.5X bead clean - combined all 4 tubes into 1 tube/pool (120ul beads) eluted in 35ul Tris
Post Bead Clean Ran 2X Qubit and TapeStation with D1000 Kit
| Sample | Run 1 | Run 2 |
|---|---|---|
| S1 | 415.66 | |
| S2 | 106712.94 | |
| Pool 1 | 52.6 | 52.2 |
| Pool 2 | 80.0 | 79.6 |
| Pool 3 | 72.0 | 71.8 |
| Pool 4 | 78.8 | 77.6 |
| Pool 5 | 105 | 104 |
| Sample | Run 1 | Run 2 |
|---|---|---|
| S1 | 443.34 | |
| S2 | 109746.89 | |
| Pool 1 | 51.6 | 51.2 |
| Pool 2 | 74.0 | 73.6 |
| Pool 3 | 68.4 | 67.8 |
| Pool 4 | 76.0 | 75.8 |
| Pool 5 | 103 | 102 |
Ran Qubit Again on 3/2/2022
| Sample | Run 1 | Run 2 |
|---|---|---|
| S1 | 433.67 | |
| S2 | 112886 | |
| Pool 1 | 46.8 | 46.4 |
| Pool 2 | 77.2 | 77.0 |
| Pool 3 | 68.4 | 68.2 |
| Pool 4 | 73.4 | 73.0 |
| Pool 5 | 101 | 98.4 |
Written on February 24, 2022