Library prep for Group 4, which is a slightly updated selection of 1800s samples.
From Qubit + TapeStation output for 1800s re-extractions, 50368, 42137, 14399 have highest concentrations and decent length distributions.
| Catalog # | Qubit conc ng/ml | TapeStation avg length | Conc. Fmol/ul |
|---|---|---|---|
| 50368 | 135.2 ng/ul | 681 bp | 322.3 fmol/ul |
| 42137 | 54.4 ng/ul | 555 bp | 159.1 fmol/ul |
| 14399 | 42.8 ng/ul | 591 bp | 117.6 fmol/ul |
14399 has lowest concentration, so its the limiter in taking equimolar Input from each extraction.
11 ul of 14399 = 11uL * 117.6fmol/ul = 1293.6 fmol ~= 1300 fmol
So, I want to use 1300 fmol of input from all 3 samples:
50368: 1300 fmol / 322.3 fmol/ul = 4.031 = 4 ul
42137: 1300 fmol / 159.1 fmol/ul = 8.171 = 8.2 ul
So, for library prep:
| Catalog # | ul for 1300 fmol | rem. volume ul | Barcode |
|---|---|---|---|
| 50368 | 4 | 7 | 27 |
| 42137 | 8.2 | 2.8 | 28 |
| 14399 | 11 | 0 | 29 |
Followed ONT Native Barcoding 96 V14 gDNA protocol, with the following modifications:
changed input quantity for each sample (see above)
Did 3 full barcode ligation rxns, each with 3.75 ul of end-prepped DNA, then pooled.
Low on SFB, so washed beads during barcode clean with fresh 80% ethanol
Qubit:
Standard 1: 38.87
Standard 2: 16411.03
G4L1 barcodes: 34.0 ng/uL
G4L1 : 59.2 ng/uL