Now that I’m satisfied with the tweaks to the Native Barcoding gDNA protocol I’ve made, I will incorporate them all for a library prep of Group 1 (Library 4) that will hopefully be good enough to sequence on MinION.
As a reminder, I’m using Native Barcoding 96 V14, gDNA protocol, with the following modifications:
DNA Repair & End-Prep
Step 4: aliquot 1000ng DNA per sample (not 400ng)
Step 6: exclude DCS, making up remaining 1uL with sample DNA or nuclease-free water
Native Barcode Ligation
Step 7: use 3.75uL end-prepped DNA for each sample, and exclude the nuclease-free water
| Catalog # | Conc. (ng/uL) | uL for 1000ng | uL for 12uL | Label/Barcode |
|---|---|---|---|---|
| 1606826 | 408 | 2.5 | 9.5 | 12 |
| 1740336 | 368 | 2.7 | 9.3 | 13 |
| 1740363 | 343 | 2.9 | 9.1 | 14 |
End-Prep Master Mix:
| 1X | 4X | |
|---|---|---|
| NEBNext FFPE DNA Repair Buffer | 0.875 uL | 3.5 uL |
| Ultra II End-Prep Reaction Buffer | 0.875 uL | 3.5 uL |
| Ultra II End-Prep Enzyme Mix | 0.75 uL | 3 uL |
| NEBNext FFPE DNA Repair Mix | 0.5 uL | 2 uL |
Made a 4X Master Mix and added 3uL to each sample
Qubit 1 (Barcodes ligated, in 35uL)
Standard 1: 38.64
Standard 2: 16901.25
Library 2: 14.9 ng/uL
Qubit 2 (Adapters ligated, in 15uL)
Standard 1: 39.66
Standard 2: 10449.45
Library 2: 36.6 ng/uL