Femto Pulse – Genomic DNA 165 kb (FP‑1002‑22, 22 cm)

Materials and equipment

Instrument & software

• Agilent Femto Pulse with FP 12‑Capillary Array, 22 cm (Part A1600‑1250‑2240).
• Femto Pulse controller software and ProSize analysis software.

Agilent Genomic DNA 165 kb kit (FP‑1002‑0275) – key components

• FP Large DNA Separation Gel
• FP Intercalating Dye
• FP gDNA Diluent Marker (DM)
• FP 165 kb Ladder
• 5× Capillary Conditioning Solution; Capillary Storage Solution
• 0.25× TE Rinse Buffer; Dilution Buffer (0.25× TE)
• BF‑P25 Blank Solution
• Eppendorf LoBind 0.5 mL tubes + wide‑bore genomic tips (included in kit) (Use only recommended/approved reagents/consumables.) ([Agilent][1])

Plates & consumables (not supplied)

• 96‑well sample plate: Eppendorf 96‑Well twin.tec semi‑skirted (#951020303) or another plate from the Agilent approved list.
• Buffer/Waste deep‑well plate: Fisher 12‑566‑120 (31 mm height).
• Adhesive PCR plate seal; multichannel pipettes; centrifuge for 96‑well plates; sub‑micron DI water. ([Agilent][2])

Specifications and acceptance targets (fast method)

• Sizing range: 1.3 kb–165 kb
• Run time: ~70 min (fast); 180 min for extended method
• Samples/run: 11 + 1 ladder (12‑capillary)
• Sample volume required: 2 µL (mixed into well with DM)
• Optimal peak signal: ~100–5,000 RFU; do not exceed 60,000 RFU
• Input gDNA concentration (total): typically 5–500 pg/µL for smears; 0.3–30 pg/µL for fragments; maximum gDNA 500 pg/µL.

Note on dilution math: Each sample well is prepared as 2 µL DNA + 18 µL Diluent Marker (10× dilution in‑well). If your tube reads 500 pg/µL, the final in‑well concentration will be ~50 pg/µL. Adjust the tube concentration beforehand if needed to stay within the kit’s recommended ranges and to avoid >60,000 RFU.

Pre‑run preparation (daily)

1. Equilibrate reagents to room temp (19–25 °C) for ≥30 min. Keep reagents at RT during sample setup.

2. Prepare gel + dye (mix gently; avoid bubbles):

   # samples	Intercalating dye	Separation gel
   12	1.0 µL	10 mL
   24	2.0 µL	20 mL
   36	3.0 µL	30 mL
   48	4.0 µL	40 mL
   96	8.0 µL	80 mL
   (Invert gently 5–10×; do not vortex.)

3. Dilute 5× Conditioning Solution to 1× with DI water (volumes: 10, 20, 30, 40, 80 mL for 12–96 samples).

4. Daily 20‑min array conditioning (recommended): In the Operation tab, add “20 min Conditioning” to the queue and run it. Ensure ≥25 mL of 1× Conditioning Solution and ≥10 mL of FP Large DNA Separation Gel are loaded.

If ladder peak is weak or missing or you observe loss of HMW peak shape after multiple runs, perform Method D Flush (0.5 N NaOH) per Agilent’s procedure before running samples.

Ladder handling and working solution (prepare fresh each day)

1. Aliquot the 165 kb ladder to minimize freeze–thaws (e.g., 7 × 5 µL in LoBind tubes). Do not vortex; mix gently with wide‑bore tips; equilibrate to RT ~30 min.

2. Prepare ladder working solution (LWS):

   • 9 µL Dilution Buffer (0.25× TE) + 90 µL Diluent Marker; vortex to mix.
   • Add 1 µL ladder aliquot, mix slowly with wide‑bore tip.
   • Final volume = 100 µL; use 20 µL per run; use within 1 day.
   • Load 20 µL LWS into well 12 of each row used.

Sample preparation (starting from Qubit‑quantified tube)

1. Record the Qubit HS concentration (ng/µL). Convert to pg/µL (1 ng/µL = 1,000 pg/µL).

2. Normalize the tube to within kit specs using 1× TE if needed:

   • For HMW smears: typically 5–500 pg/µL.
   • For distinct fragments: 0.3–30 pg/µL.
   • Never exceed 500 pg/µL in the tube.

3. On a 96‑well sample plate, dispense 18 µL Diluent Marker into each sample well (fill unused wells in the row with 20 µL BF‑P25 Blank).

4. Add 2 µL of each gDNA sample into the 18 µL DM (final 20 µL per well). Mix gently 2–3× with a wide‑bore tip; avoid shearing. Spin the plate briefly to clear bubbles.

5. Target signal: Adjust tube concentration so that run peaks fall ~100–5,000 RFU and <60,000 RFU. If too high, dilute tube further and repeat.

6. Run immediately. If delayed, seal and store 2–8 °C and analyze same day (or overlay with mineral oil per instrument manual guidance).

Instrument setup and run

1. Load plates (front drawers):

   • Buffer (B): place 1× Inlet Buffer plate (1.0 mL/well) in row A; replace daily.
   • Waste (W): deep‑well plate as waste.
   • Rinse (M): 0.25× TE Rinse Buffer, 200 µL/well, row A; replace daily.
   • Storage: place Capillary Storage Solution plate (1.0 mL/well, row H) and replace every 2–4 weeks.
   • Sample: place prepared sample plate (wells with 20 µL; well 12 contains ladder LWS).

2. In Femto Pulse software → Operation tab:

   • Select the tray/row.

   • Enter Sample IDs (optional: Tray Name, Folder Prefix, Notes).

   • Add to Queue and choose the method based on capillary length:

     • FP‑1002‑22 — gDNA 165 kb (fast)
     • FP‑1002E‑22 — Extended gDNA 165 kb (use if very large DNA compresses at end of fast run).

   • Start the separation.

Data analysis & acceptance criteria (ProSize)

• Confirm the 165 kb ladder pattern and that the 165 kb peak is present with adequate signal.
• For samples, evaluate smear peak distribution and max RFU; re‑run highly saturated samples with lower input.
• If you observe a narrow, high‑MW peak at the end of the fast run, repeat with the Extended method (FP‑1002E‑22) for better resolution.

Post‑run

• If multiple runs are planned the same day, continue with routine operation.
• If ladder signal declines or 165 kb peak degrades over time, execute Method D Flush (0.5 N NaOH → 20 min) per Agilent’s instructions to restore performance; then resume.
• For longer idle periods, follow the instrument manual for capillary array wet storage and shutdown. ([Agilent][2])

Troubleshooting (quick pointers)

• Ladder <1,000 RFU or absent → Perform Method D Flush; verify ladder handling (no vortexing; wide‑bore tips; fresh aliquot). ([Agilent][1])
• Sample >60,000 RFU → Tube concentration too high; dilute with 1× TE, re‑prepare well, and re‑run. ([Agilent][1])
• No sample signal (LM only) → Input below detection, sample not added, or wrong row selected. Re‑check plate and concentrations. ([Agilent][1])

Appendix A — Quick volume tables (from Agilent quick guide)

Gel + dye and 1× Conditioning Solution quantities are listed above (“Pre‑run preparation”). Minimum well volumes: instrument requires ≥20 µL in each sample well for proper injection. ([Agilent][2])

References (Agilent documents)

• Genomic DNA 165 kb Kit – Quick Guide (latest): analytical specs; plate prep; sample DM mix 2 µL + 18 µL; ladder handling; daily conditioning; Method D Flush.
• Femto Pulse System User Manual (latest): instrument drawers; minimum well volumes; approved plates; run controls; storage. ([Agilent][2])
• Genomic DNA 165 kb kit – Method files page (Agilent): Method D Flush & 20‑min conditioning notes. ([Agilent][3])

One‑page “at‑the‑bench” checklist

• Reagents at RT ≥30 min; gel+dye mixed; 1× conditioning ready.
• Daily 20‑min conditioning run completed.
• Ladder aliquot thawed/mixed with wide‑bore tips; LWS prepared; 20 µL in well 12.
• Tube conc (from Qubit) adjusted to kit range; 2 µL sample + 18 µL DM per well; brief spin; no bubbles.
• Load Buffer (B), Waste (W), Rinse (M), Storage, and Sample plates as specified.
• In software: select tray/row → FP‑1002‑22 method → Start. Extended method if high‑end compression seen.
• Ladder looks correct; sample RFUs 100–5,000 (<<60,000). If not, dilute and re‑run.