Fragment Analyzer – HS Large Fragment 50Kb

Method: DNF-464-33 - HS Large Fragment 50Kb.mthds

Instrument: Agilent/Advanced Analytical Fragment Analyzer (33 cm, 10-capillary array)

Analysis Mode: NGS

1. Document Control

• SOP ID: FA-HSLF-50Kb-33cm
• Version: 1.0
• Effective Date: ☐☐☐☐-☐☐-☐☐
• Prepared by: ☐
• Approved by: ☐
• Review cycle: 12 months

2. Purpose

To define the procedure for running high-sensitivity large-fragment DNA analysis on the Fragment Analyzer using the DNF-464-33 method and a 33 cm, 10-capillary array.

3. Scope

Applies to all operators analyzing high molecular weight DNA for NGS library QC or large-fragment assessment using the specified method on a Fragment Analyzer.

4. Responsibilities

• Operator: Execute this SOP, record run metadata, verify QC, and report issues.
• Lab lead: Ensure training, instrument maintenance, and SOP review.

5. Safety

• Wear lab coat, safety glasses, and disposable gloves.
• Handle gels, buffers, and plates per SDS.
• Avoid contact with high voltage areas during operation. Only open instrument when software indicates safe.
• Dispose of consumables as per local regulations.

6. References

• Instrument user manual for the Fragment Analyzer.
• Kit instructions for HS Large Fragment 50Kb chemistry.
• Local laboratory policies for data retention and equipment maintenance.

7. Materials and Equipment

Instrument and configuration

• Fragment Analyzer with 33 cm array, 10 capillaries.
• Array serial and usage count recorded before each run.

Reagents and consumables

• HS Large Fragment 50Kb gel cartridge compatible with DNF-464-33.
• Buffer reservoirs and rinse solution as per kit.
• 96‑well plate(s) and plate seals or caps.
• DNA ladder and markers as required by the kit.
• Nuclease-free water and low-retention tips.

Software

• FA Software v1.2.0.11 or later.

8. Sample Requirements

• DNA in kit-recommended buffer.
• Avoid particulates and salts that interfere with electrokinetic injection.
• Recommended concentration and volume per kit instructions.

9. Pre‑Run Checks

1. Inspect array for leaks, kinks, or salt crystallization. Record array serial and usage count.

2. Confirm instrument details in software:

   • Device Serial: record (e.g., 4172)
   • FA Version: record (e.g., 1.2.0.11)

3. Verify consumables:

   • Correct gel loaded and Gel Selection: Gel 1.
   • Fresh buffer and rinse trays in correct positions.

4. Equilibrate reagents to room temperature if required by kit.

5. Prepare plate map with samples, ladder, controls, and blanks.

10. Plate Preparation

1. Thaw and mix reagents gently. Spin down briefly.
2. Aliquot ladder, controls, and samples into designated wells.
3. Seal the plate. Briefly centrifuge to remove bubbles. Keep sealed until loading.

11. Instrument Setup (Software)

Set parameters exactly as follows unless method validation specifies otherwise:

• Method Name: DNF-464-33 - HS Large Fragment 50Kb.mthds
• Analysis Mode: NGS
• Gel Prime: No
• Full Conditioning: Yes
• Gel Prime to Buffer: No
• Gel Selection: Gel 1
• Perform Prerun: 5.0 kV for 30 s
• Rinse: Tray 3, Row A, # Dips: 1
• Marker 1: No (unless the kit run requires an internal marker; follow kit guidance)
• Sample Injection: 5.0 kV for 30 s
• Separation: 5.0 kV for 60.0 min
• Tray Name: Tray-1
• # of Capillaries: 10

File management

• Create or select a run folder. Recommended format:

  • YYYY MM DD/HH‑MM‑SS/<auto>.raw
  • Example: C:\AATI\Data\2022 07 18\14-03-50\2022 07 18 14H 03M.raw

12. Run Procedure

1. Load the sealed sample plate into the instrument.
2. Confirm tray positions and liquid levels.
3. In software, confirm the method parameters match Section 11.
4. Start the run. Observe for the first 2–3 minutes for abnormal current, leaks, or errors.
5. Allow the 60.0 min separation to complete.

13. Post‑Run Actions

1. Save and back up all files generated by the run, including the .raw and analysis reports.
2. Export the Run Summary and electropherograms as required by your reporting template.
3. If subsequent runs are not planned the same day, follow kit guidance to store gel and perform any required rinses or conditioning before shutdown.

14. Quality Control and Acceptance

• Ladder and control wells exhibit expected profile and sizing (per kit IFU).
• Baseline is stable with distinct peaks where applicable.
• Migration time consistent across capillaries; no persistent current alarms.
• If any criterion fails, document, recondition, and repeat controls before analyzing unknowns.

15. Troubleshooting

• Weak or absent signal: Verify injection settings, sample concentration, plate seal integrity, and capillary tips in wells.
• High noise or drifting baseline: Re‑condition array, check buffer freshness and tray cleanliness.
• Inconsistent sizing: Confirm ladder integrity, plate map accuracy, and temperature stability.
• Frequent current faults: Inspect for bubbles, low liquid levels, or salt contamination. Replace consumables as needed.

16. Maintenance

• Perform Full Conditioning at the start of the day or as required by kit.
• Replace gels and buffers per shelf‑life and use counts.
• Track Array Usage Count at each run and follow vendor guidance for replacement.
• Clean trays weekly or after spills. Inspect electrodes and stage area.

17. Records to Retain

For each run record the following in the run log or ELN:

• Date and time of run.
• Operator name/initials.
• Device Serial and FA Software Version.
• Array Serial # and Usage Count.
• Method Name and all key parameters in Section 11.
• Plate map and sample IDs.
• File path to raw data and reports.
• QC outcomes and any deviations.

18. Run Log Template

B. Abbreviations

• FA: Fragment Analyzer
• HMW: High molecular weight
• IFU: Instructions for Use
• ELN: Electronic Lab Notebook