Library, prep for Group 4 Library 2, since, G4L1 had low MinION output. ONT support has also requested I try prepping and running a DNA control Sample Library for 1-2hr, to check flow cell function, so I’ll be doing 2 preps.
Following Native Barcoding 96 V14 gDNA protocol with the following modifications:
Use below input quantities
For all samples, did 3 full barcode ligation rxns before pooling.
Washed beads during barcode clean with 80% ethanol (fresh)
DCS Library
Use Full 12uL of diluted DCS as input. DCS is 10ng/uL so diluted DCS is 3.3ng/uL. Will use Barcode 33.
G4L2
| Catalog # | [dsDNA] | volume left | length | ng left | fmol left |
|---|---|---|---|---|---|
| 50368 | 135.2 ng/uL | 15uL | 681 | 2028ng | 4834 fmol |
| 42137 | 54.4 ng/uL | 9.5uL | 555 | 516.8ng | 1512 fmol |
| 14399 | 42.8 ng/uL | 6.5uL | 591 | 278.2ng | 764.2 fmol |
So limiting, extraction is 14399. Will use ~800 fmol from all extractions.
| Catalog # | ng for. 800fmol | uL for 200fmol | uL for 11uL | Barcode |
|---|---|---|---|---|
| 50368 | 335.6 ng | ~2.5uL | 8.5 | 30 |
| 42137 | 273.5 ng | ~5 uL | 6 | 31 |
| 14399 | 291.2 ng | 6.5uL | 4.5uL | 32 |
End-repair, Master Mix (5x)
FFPE Buffer: 4.375 uL
Ultra II Buffer: 4.375 uL
Ultra II Mix: 3.75 uL
FFPE Mix: 2.5 uL
Qubits:
Standard 1: 48.12
Standard 2: 15478.64
DCS barcode: 0.528 ng/uL
DCS library: 0.776 ng/uL
G4L2 barcode: 11.1 ng/uL
G4L2 library: 22.0 ng/uL