pFC9 EcoRI SacI digestion, LF extraction and cleanup #
Now that I am somewhat mistrustful of existing pFC9 LF for future Gibson assemblies I am making more fragment today hopefully for use on Sunday / Monday.
pFC9 EcoRI SacI digestion #
Digestion reagents
- 3.3 ul (1 ug) pFC9
- 2 ul 10x CutSmart buffer
- 1 ul EcoRI-HF
- 1 ul SacI-HF
- 12.7 ul npH20
Incubate in hot room 37C for 1.5 hours.
Quintara submission #
Given yesterday’s sequencing results indicating that the 9/8/21 Gibson assembly had some assembled plasmids (although inexplicably included VR-3) I am submitting 2 of each of the rest of the insert colonies for sequencing. Reaction layout is at the Quintara spreadsheet table 9-20-21 and shown in the table below.
| Plasmid | Plasmid volume (ul) | H20 | Forward primer volume (ul) | Real concentration | Total volume | Label |
|---|---|---|---|---|---|---|
| VR-17-2 | 12.5 | 0 | 2.5 | 67.92 | 15 | 1 |
| VR-17-3 | 12.5 | 0 | 2.5 | 44.17 | 15 | 2 |
| VR-20-1 | 10.15228426 | 2.35 | 2.5 | 79.99 | 15.00228426 | 3 |
| VR-20-2 | 12.5 | 0 | 2.5 | 44.58 | 15 | 4 |
| VR-21-1 | 12.5 | 0 | 2.5 | 49.92 | 15 | 5 |
| VR-21-2 | 12.5 | 0 | 2.5 | 52.42 | 15 | 6 |
| VR-22-1 | 11.62790698 | 0.87 | 2.5 | 80.01 | 14.99790698 | 7 |
| VR-22-2 | 12.5 | 0 | 2.5 | 50.92 | 15 | 8 |
| VR-25-1 | 12.5 | 0 | 2.5 | 64.5 | 15 | 9 |
| VR-25-2 | 12.5 | 0 | 2.5 | 72.92 | 15 | 10 |
| VR-26-1 | 12.5 | 0 | 2.5 | 80 | 15 | 11 |