Plasmid dilution and primer prep — sequencing sent off

Contributors: Esther, Tank

Plasmid dilutions and primer prep (p. 165)

Nine samples diluted to a common target for sequencing. Sample 9’s plasmid concentration (145.6 ng/µl) matches the colony-3 miniprep from 22 September exactly, so that’s very likely the same prep carried through to here — but which of samples 1–7 correspond to which named colony (CO2, CO4, CO7, CO12, T4, T14, T15, β-glucosidase, etc. from the 16 September miniprep list) isn’t established in the notebook itself. Several values are overwritten, and sample 8 is struck out throughout both passes of the table.

Target: 40 µl final volume per sample, two different final concentrations recorded (30 ng/µl and 26.69 ng/µl) without a clear note of which sample got which.

Row 5’s final volume looks like it may read “46” rather than “40” in the original — flagged rather than silently corrected to match the other rows.

A second pass of the same table appears directly below it in the notebook, largely repeating the first with some values overwritten (e.g. sample 1’s plasmid volume corrected from 4.00 to ~4.0, sample 7’s from 4.716 to 4.71) and the final-concentration values for samples 6 and 7 swapped relative to the first pass (30 ↔ 26.69) — reproduced above as the first pass; the second pass isn’t transcribed separately given the overlap.

Primers (100 µM stock)

• Start-For HX2: 100 µM × V₁ = 3.2 µM × 20 µl
• End-Rev1: 100 µM × V₁ = 3.2 µM × 20 µl
• Working mix: 6.4 µl primer + 13.6 µl NFW (an earlier, struck-out calculation gave 0.64 µl primer + 14.36 µl NFW instead)

Final dilution volumes (all ticked off)

A small note in the right margin next to samples 4–5 is illegible — possibly referencing other sample labels, but not confident enough in the reading to transcribe it.

Sequencing sent off this day.