PCR Product Cleanup Protocol

Protocol Overview

Purpose: Remove residual primers and unincorporated dNTPs from PCR products to optimize Sanger sequencing quality

Method: Enzymatic degradation using ExoCleanUp FAST (HL-Exonuclease I + rSAP)

Time Required: 8-10 minutes per batch

Kit: VWR ExoCleanUp FAST (Cat. No. 733-2593, 500 reactions)

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Learning Objectives

By completing this protocol, you will:

• Execute enzymatic PCR cleanup for Sanger sequencing preparation
• Understand why residual primers and dNTPs interfere with sequencing
• Compare enzymatic vs. physical cleanup methods
• Prepare sequencing-ready samples with proper labeling and documentation
• Calculate cost-benefit analysis for different cleanup approaches

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Safety Information

Hazard Classification

According to the Safety Data Sheet (SDS_ExoCleanUp_VWRC733-2593.pdf):

• Classification: Non-hazardous according to Regulation (EC) No 1272/2008 [CLP]
• Labeling: No hazard pictograms required
• Components: Recombinant enzymes (HL-ExoI, rSAP) in buffered aqueous solution

Personal Protective Equipment

Required:

• Safety glasses or face shield
• Laboratory coat
• Nitrile gloves
• Closed-toe shoes

Optional: None required for this protocol

Emergency Procedures

Skin contact: Wash with soap and water; not expected to cause harm

Eye contact: Rinse with water for 15 minutes; consult physician if irritation persists

Ingestion: Rinse mouth with water; seek medical attention

Spills: Wipe with absorbent material; dispose as biological waste

Emergency contact: UC Riverside Environmental Health & Safety (951) 827-5528

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Materials and Reagents

ExoCleanUp FAST Kit Components

From VWR (Cat. No. 733-2593):

• ExoCleanUp FAST enzyme mix (1 mL, green cap)
• Contains: Heat-labile Exonuclease I (HL-ExoI)
• Contains: Recombinant Shrimp Alkaline Phosphatase (rSAP)
• Storage: -20°C
• Color: Colorless liquid
• Stability: Stable for 12 months at -20°C; avoid >5 freeze-thaw cycles

Equipment Required

• Thermal cycler or heat block (37°C and 80°C capability)
• Micropipettes: P2 or P10 (for 2 µL additions)
• PCR tubes (0.2 mL, thin-walled) or 96-well PCR plate
• Ice bucket with ice
• Tube rack
• Vortex mixer (optional, for mixing)
• Microcentrifuge (optional, for brief spin-down)

Additional Materials

• PCR products from Module 7 (COI amplification)
• Nuclease-free water (for volume adjustments if needed)
• Permanent marker for tube labeling
• Agarose gel supplies (for quality control verification)

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Pre-Lab Preparation

1. Retrieve PCR Products

From Module 7, you should have:

• Sample A: -80°C frozen mosquito + column extraction + COI PCR
• Sample B: 95% ethanol mosquito + column extraction + COI PCR
• Sample C: Silica gel mosquito + column extraction + COI PCR
• Positive control: Known arthropod DNA + COI PCR
• Negative control: No template control (should be negative)

Action: Retrieve PCR products from 4°C or -20°C storage; thaw on ice if frozen

2. Verify PCR Success (Optional but Recommended)

Run 5 µL of each PCR product on 1.5% agarose gel:

• Expected band: ~710 bp (COI amplicon)
• Intensity: Bright, clear band
• Negative control: No band

Decision point: Only clean up samples with successful amplification (clear band at expected size)

3. Prepare Thermal Cycler

Program thermal cycler with ExoCleanUp protocol:

```

Step 1: 37°C for 5 minutes (digestion)

Step 2: 80°C for 5 minutes (inactivation)

Step 3: 4°C hold (storage)

```

Note: Minimum times are 2 minutes (37°C) and 3 minutes (80°C); we use 5 minutes for safety margin

4. Thaw ExoCleanUp FAST

Remove ExoCleanUp FAST from -20°C freezer:

• Place immediately on ice
• Allow to thaw completely (~5 minutes)
• Mix gently by pipetting or light vortex
• Keep on ice at all times during use

Critical: Never leave ExoCleanUp FAST at room temperature; enzyme activity decreases with warming

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Step-by-Step Protocol

Step 1: Reaction Setup (2 minutes)

For each PCR product to be cleaned:

1. Label clean PCR tubes or use existing PCR tubes
2. Write sample ID clearly
3. Add "-CU" suffix (e.g., "Sample A-CU" for "cleaned-up")

1. Transfer or keep 5 µL of PCR product in tube
2. If PCR performed in 25 µL volume, use 5 µL for cleanup
3. Remaining 20 µL can be saved at -20°C as backup

1. Add 2 µL ExoCleanUp FAST directly to PCR product
2. Pipette slowly and carefully (viscous solution)
3. Dispense enzyme into liquid, not on tube wall
4. Keep ExoCleanUp FAST on ice between samples

1. Mix reaction thoroughly
2. Option A: Pipette up and down 5-8 times
3. Option B: Vortex briefly (1-2 seconds) then pulse spin
4. Ensure complete mixing (enzyme must contact primers/dNTPs)

Volumes:

• PCR product: 5 µL
• ExoCleanUp FAST: 2 µL
• Total reaction volume: 7 µL

Scaling: For larger PCR volumes, scale proportionally:

• 10 µL PCR product → 4 µL ExoCleanUp FAST
• 25 µL PCR product → 10 µL ExoCleanUp FAST
• Ratio is always 2:5 (enzyme:PCR product)

Step 2: Digestion (5 minutes at 37°C)

1. Place tubes in thermal cycler preheated to 37°C
2. Close lid securely (heated lid should be 40-45°C)
3. Start timer for 5 minutes

1. During incubation:
2. HL-ExoI degrades single-stranded primers (3' to 5' direction)
3. rSAP dephosphorylates unincorporated dNTPs
4. Double-stranded PCR products remain intact

Timing considerations:

• Minimum: 2 minutes (sufficient for typical 25-mer primers)
• Standard: 5 minutes (recommended for all applications)
• Extended: Up to 15 minutes (does not harm amplicons)

Troubleshooting: If previous cleanups showed residual primers (poor sequencing), extend to 10 minutes

Step 3: Enzyme Inactivation (5 minutes at 80°C)

1. Thermal cycler automatically proceeds to 80°C
2. Verify temperature reached 80°C (check display)
3. Hold for 5 minutes

1. During inactivation:
2. HL-ExoI denatures rapidly (t½ < 1 minute at 80°C)
3. rSAP inactivates (t½ ~ 2 minutes at 80°C)
4. DNA remains stable (no denaturation at 80°C)

Timing considerations:

• Minimum: 3 minutes (>99.9% inactivation)
• Standard: 5 minutes (recommended)
• Extended: 10 minutes (for long-term storage before sequencing)

Temperature critical: Ensure block reaches 80°C; lower temperature reduces inactivation efficiency

Step 4: Cooling and Storage (2 minutes)

1. Allow thermal cycler to cool to 4°C (or remove tubes)
2. Automatic hold at 4°C if programmed
3. Or remove tubes when temperature drops below 40°C

1. Cleaned PCR products can be:
2. Used immediately for sequencing (proceed to Step 5)
3. Stored at 4°C for up to 1 week
4. Stored at -20°C indefinitely (long-term storage)

Quality check: Cleaned products should look identical to pre-cleanup (clear, no precipitate)

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Quality Control: Confirming Successful Cleanup

Method 1: Gel Electrophoresis Comparison (Recommended)

Purpose: Verify primers removed, DNA intact

Protocol:

1. Prepare 1.5% agarose gel with ethidium bromide or SYBR Safe
2. Load gel lanes:
3. Lane 1: 1 kb DNA ladder
4. Lane 2: 3 µL raw PCR product (before cleanup)
5. Lane 3: 3 µL cleaned PCR product (after cleanup)
6. Lane 4: Empty
7. Repeat for each sample

1. Run gel at 100V for 30 minutes
2. Image on UV transilluminator

Expected results:

• Raw PCR product: Bright band at 710 bp + possible faint primer band at 25 bp
• Cleaned PCR product: Bright band at 710 bp, no primer band
• Intensity: Cleaned band should match raw band (no DNA loss)

Interpretation:

• ✓ Success: 710 bp band present, no primer band, similar intensity
• ✗ Incomplete cleanup: Primer band still visible (extend digestion time)
• ✗ DNA degradation: Smearing or reduced intensity (check inactivation temperature)

Method 2: Spectrophotometry (Optional)

Using NanoDrop:

1. Measure 1 µL cleaned PCR product
2. Record concentration and ratios:
3. Concentration: Should match pre-cleanup (±10%)
4. 260/280 ratio: Should be 1.8-2.0 (pure DNA)
5. 260/230 ratio: Should be 2.0-2.2 (minimal contamination)

Interpretation: Ratios should not change significantly; concentration should remain stable

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Preparation for Sanger Sequencing

Sample Concentration Requirements

UCR Genomics Core Facility requirements (verify with current submission guidelines):

• PCR product concentration: 10-50 ng/µL (optimal: 20-30 ng/µL)
• PCR product size: 300-1000 bp (your COI product is 710 bp - perfect)
• Volume submitted: 12 µL (10 µL for reaction + 2 µL dead volume)
• Primer concentration: 3.2 µM (5 µL of sequencing primer)

Concentration Adjustment (if needed)

If concentration too high (>50 ng/µL):

• Dilute with nuclease-free water to 20-30 ng/µL
• Calculate dilution: C₁V₁ = C₂V₂
• Example: 80 ng/µL stock → 25 ng/µL working
• 80 × V₁ = 25 × 20 µL
• V₁ = 6.25 µL (stock) + 13.75 µL (water) = 20 µL final

If concentration too low (<10 ng/µL):

• Use larger volume (up to 20 µL) in sequencing reaction
• Or concentrate using spin column or vacuum concentration
• Or repeat PCR with more cycles (35-40 instead of 30-32)

If concentration perfect (10-50 ng/µL):

• Use directly without dilution
• This is ideal scenario

Sequencing Primer Information

For COI barcoding, you will sequence with:

• Forward sequencing primer: LCO1490 (same as PCR forward primer)
• Sequence: 5'-GGTCAACAAATCATAAAGATATTGG-3'
• Reverse sequencing primer: HCO2198 (same as PCR reverse primer)
• Sequence: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3'

Recommendation: Sequence both directions (forward and reverse) for maximum coverage and accuracy

• Forward read: 700-900 bp from 5' end
• Reverse read: 700-900 bp from 3' end
• Overlap: ~400-500 bp (for quality verification)
• Total coverage: Entire 710 bp amplicon

Sample Submission Checklist

Before submission, verify:

• Gel image shows successful PCR amplification (~710 bp band)
• Cleaned PCR product concentration measured (10-50 ng/µL ideal)
• Volume sufficient for sequencing (≥12 µL per reaction)
• Tubes clearly labeled with sample ID
• Sequencing primers prepared at 3.2 µM concentration
• Submission form completed with sample details
• Budget/billing information provided

Submit to: UC Riverside Genomics Core Facility

• Location: Genomics Building, Room 1208
• Contact: genomics.core@ucr.edu
• Turnaround: 24-48 hours for standard Sanger sequencing
• Cost: ~$6 per sample per direction (verify current pricing)

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Troubleshooting Guide

Problem: Poor Sequencing Quality (Short Reads, Low Quality Scores)

Symptoms:

• Read length <400 bp (should be 700-900 bp)
• Phred quality scores 20% of bases
• Mixed bases (N calls) throughout sequence

Possible causes and solutions:

1. Incomplete primer degradation
2. Cause: Insufficient ExoCleanUp digestion time
3. Solution: Extend 37°C incubation from 5 to 10 minutes in future reactions

1. Residual active enzymes
2. Cause: Incomplete inactivation at 80°C
3. Solution: Extend 80°C inactivation from 5 to 10 minutes; verify thermal cycler temperature calibration

1. PCR product concentration too low
2. Cause: Dilute template submitted for sequencing
3. Solution: Measure concentration; must be >10 ng/µL; concentrate if needed

1. Sequencing primer degraded or incorrect concentration
2. Cause: Old primer stock or dilution error
3. Solution: Prepare fresh primers at 3.2 µM; verify concentration

Problem: No Sequencing Data Obtained

Symptoms:

• Sequencing facility reports "no data" or "failed reaction"
• Chromatogram shows flat baseline with no peaks

Possible causes and solutions:

1. No PCR product in tube
2. Cause: PCR failed or wrong tube submitted
3. Solution: Verify band on gel before cleanup; always run QC gel

1. PCR product concentration too low
2. Cause: <5 ng/µL submitted
3. Solution: Measure concentration with Qubit or NanoDrop; must be >10 ng/µL

1. Wrong sequencing primer submitted
2. Cause: Primer mismatch to amplicon
3. Solution: Verify primer sequences match PCR primers used

1. Tube labeling error
2. Cause: Sample ID confusion during submission
3. Solution: Cross-reference submission form with tube labels

Problem: DNA Degradation During Cleanup

Symptoms:

• Gel shows smearing instead of discrete band
• Concentration drops >20% after cleanup
• Sequencing quality poor or failed

Possible causes and solutions:

1. Over-digestion with ExoCleanUp
2. Cause: Extended incubation with active enzymes
3. Solution: Reduce 37°C digestion time to 2-3 minutes minimum

1. Prolonged high temperature exposure
2. Cause: Extended 80°C inactivation (>15 minutes)
3. Solution: Use standard 5-minute inactivation; do not exceed 10 minutes

1. Contaminated ExoCleanUp enzyme
2. Cause: Non-specific nuclease contamination
3. Solution: Use fresh aliquot; avoid repeated freeze-thaw; keep on ice

Problem: Inconsistent Results Between Samples

Symptoms:

• Some samples sequence well, others fail
• Variable cleanup efficiency across batch

Possible causes and solutions:

1. Temperature variation in thermal cycler
2. Cause: Edge wells cooler than center wells
3. Solution: Use only center wells; verify block calibration; increase incubation times by 2 minutes

1. Pipetting errors during setup
2. Cause: Variable ExoCleanUp volume added
3. Solution: Use calibrated pipettes; pipette carefully with viscous enzyme solution; change tips between samples

1. Different PCR conditions produced variable products
2. Cause: Some samples have higher primer concentration or primer dimers
3. Solution: Standardize PCR setup; use master mix; verify all PCR reactions comparable before cleanup

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Data Recording and Analysis

Record in Lab Notebook

For each sample, document:

Pre-cleanup:

• Sample ID
• PCR product concentration (from Qubit/NanoDrop)
• PCR product volume used for cleanup
• Gel image (band intensity, size)

During cleanup:

• Date and time
• ExoCleanUp FAST lot number
• Digestion time used (37°C)
• Inactivation time used (80°C)
• Any deviations from protocol

Post-cleanup:

• Cleaned product concentration (if measured)
• Gel image comparison (before/after cleanup)
• Volume remaining for sequencing submission
• Sequencing submission date and sample ID

Cost Analysis

Calculate per-sample costs:

ExoCleanUp FAST method:

• Enzyme cost: $0.24 per reaction (500-reaction kit at $120)
• Labor: 1 minute per sample (setup time)
• Equipment: Thermal cycler (already available)
• Total: ~$0.24 per sample

Spin column method (for comparison):

• Column cost: $2.00 per reaction (typical commercial kit)
• Labor: 10 minutes per sample (multiple pipetting steps)
• Equipment: Centrifuge
• Consumables: Extra tubes
• Total: ~$2.10 per sample

Magnetic bead method (for comparison):

• Bead cost: $0.10 per reaction (homemade SPRI beads)
• Labor: 15 minutes per sample (multiple wash steps)
• Equipment: Magnetic separation rack
• Consumables: Fresh 80% ethanol, extra tubes
• Total: ~$0.30 per sample + labor

Analysis: ExoCleanUp FAST provides 8-9× cost savings over spin columns with 10× faster processing time

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Protocol Modifications for Special Cases

High-Throughput 96-Well Format

For processing many samples simultaneously:

1. Use 96-well PCR plate instead of individual tubes
2. Transfer 5 µL PCR product to each well
3. Add 2 µL ExoCleanUp FAST using 12-channel pipette

1. Seal plate with adhesive PCR film or heated cap mat
2. Ensure complete seal to prevent evaporation
3. Use plate compression device if available

1. Incubate in plate-format thermal cycler
2. Program identical to tube protocol
3. Verify thermal uniformity across plate

1. Process time: ~10 minutes for entire plate (96 samples)

Cleanup of Large PCR Products (>1 kb)

For amplicons larger than 1 kb:

• Use same protocol but extend digestion time to 10 minutes
• Primers longer than 25 bp require more time for complete degradation
• Verify complete cleanup by gel electrophoresis

Cleanup of Low-Concentration PCR Products

If PCR product <10 ng/µL:

• Use entire PCR volume (all 25 µL) for cleanup
• Add 10 µL ExoCleanUp FAST (maintain 2:5 ratio)
• Final volume: 35 µL
• May need to concentrate after cleanup using spin column or vacuum centrifugation

Cleanup for Applications Other Than Sequencing

ExoCleanUp FAST also works for:

• SNP genotyping: Removes dNTPs that compete with ddNTPs in primer extension
• Microarray hybridization: Removes primers that could bind non-specifically to array probes
• Pyrosequencing: Removes primers and dNTPs before template preparation

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Post-Lab Cleanup and Waste Disposal

Cleanup Procedures

1. Turn off thermal cycler after completing protocol
2. Store ExoCleanUp FAST immediately at -20°C
3. Do not leave at room temperature
4. Mark tube with date if first use
5. Dispose of pipette tips in biological waste
6. Clean work area with 70% ethanol

Waste Disposal

Biological waste:

• Used PCR tubes containing cleaned products
• Pipette tips
• Gloves
• Dispose in biohazard waste containers

No special chemical waste generated:

• ExoCleanUp FAST classified as non-hazardous
• Reaction products are inactive enzymes in buffer
• Standard biological waste disposal appropriate

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Assessment and Evaluation

Success Criteria

Your PCR cleanup is successful if:

• Gel shows 710 bp band with no primer band
• Band intensity unchanged from pre-cleanup
• Sequencing submission requirements met (concentration, volume)
• Protocol completed within 15 minutes (including QC gel)

Common Mistakes to Avoid

1. Not keeping ExoCleanUp FAST on ice - reduces enzyme activity
2. Insufficient mixing - enzyme must contact primers/dNTPs
3. Wrong volume ratio - must maintain 2:5 enzyme:PCR product ratio
4. Incomplete inactivation - must reach 80°C for full 5 minutes
5. Submitting uncleaned PCR products - sequencing will fail or be poor quality

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Appendix: Comparison with Alternative Methods

ExoCleanUp FAST vs. ExoSAP-IT

Both use enzymatic degradation:

ExoCleanUp FAST:

• Heat-labile exonuclease (80°C inactivation)
• 5-minute protocol
• Lower cost per reaction
• Gentler on DNA

ExoSAP-IT:

• Traditional exonuclease (85°C inactivation)
• 15-minute protocol
• Higher cost per reaction
• Longer heat exposure

Recommendation: ExoCleanUp FAST preferred for routine Sanger sequencing

ExoCleanUp FAST vs. Spin Columns

When to use ExoCleanUp FAST:

• Sanger sequencing preparation
• Cost-sensitive applications
• Time-critical turnaround
• Small sample numbers (<100)

When to use spin columns:

• Comprehensive contaminant removal needed (salts, proteins, enzymes)
• Buffer exchange required
• Cloning applications (removing PCR enzymes before transformation)
• Very short amplicons (<100 bp) that need concentration

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References and Further Reading

1. ExoCleanUp FAST Product Information
2. VWR Cat. No. 733-2593 Protocol Sheet (10/2019)
3. SDS: Safety Data Sheet Rev. 7.5 (09.01.2025)

1. Enzyme Mechanisms
2. Lehman, I. R. (1960). The deoxyribonucleases of Escherichia coli. Journal of Biological Chemistry, 235(5), 1479-1487.
3. Weiss, B., & Richardson, C. C. (1967). Enzymatic breakage and joining of deoxyribonucleic acid. Journal of Biological Chemistry, 242(19), 4270-4283.

1. PCR Cleanup Methods Comparison
2. Rosenthal, A., & Charnock-Jones, D. S. (1992). New protocols for DNA sequencing with dye terminators. DNA Sequence, 3(2), 61-64.
3. Werle, E., Schneider, C., Renner, M., Völker, M., & Fiehn, W. (1994). Convenient single-step, one tube purification of PCR products for direct sequencing. Nucleic Acids Research, 22(20), 4354-4355.

1. Sanger Sequencing Quality Optimization
2. Sanger, F., Nicklen, S., & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, 74(12), 5463-5467.
3. Shendure, J., & Ji, H. (2008). Next-generation DNA sequencing. Nature Biotechnology, 26(10), 1135-1145.

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Protocol prepared for ENTM201L - Molecular Entomology: DNA Barcoding Laboratory

UC Riverside, Department of Entomology

Fall 2025