Experiment

Gel Extraction Using PureLink™ Quick Gel Extraction Kit

Materials Supplied in the Kit

• Gel Solubilization Buffer (L3)
• Binding Buffer (B2)
• Wash Buffer (W1)
• Elution Buffer (E1; 10 mM Tris-HCl, pH 8.5)
• PureLink™ Clean-up Spin Columns in Wash Tubes
• PureLink™ Elution Tubes

Materials You Need to Provide

• Agarose gel with DNA fragment of interest
• Clean razor blade and weigh paper/tray
• Scale (sensitive to 0.001 g)
• Water bath or heat block set at 50 °C
• Microcentrifuge (>10,000 × g)
• DNase-free pipettes and tips
• 96–100% ethanol
• (Optional) 100% isopropanol

---

Buffer Preparation (First Use Only)

1. Add 10 mL 100% isopropanol to Binding Buffer (B2).
2. Add 64 mL 96–100% ethanol to Wash Buffer (W1).
3. Mark buffer labels to indicate additions. Store at room temperature.

---

Step-by-Step Protocol

1. Excise the DNA Band

1. Run DNA on agarose gel until the band of interest is clearly separated.
2. Wearing gloves, cut out the DNA band with a clean razor blade — minimize surrounding agarose.
3. Place the gel slice into a microcentrifuge tube (≤400 mg per tube). If >400 mg, divide into smaller pieces.
4. Weigh the tube and record the gel slice weight.

2. Dissolve the Gel

1. Add Gel Solubilization Buffer (L3):
   • ≤2% agarose: 3 volumes buffer per 1 volume gel
   • 2% agarose: 6 volumes buffer per 1 volume gel (Example: 1.2 mL buffer for 400 mg gel at ≤2% agarose)
2. Incubate at 50 °C for ≥10 min, mixing every 3 min until fully dissolved.
3. After complete dissolution, incubate an additional 5 min.
4. (Optional) Add 1 gel volume of 100% isopropanol to improve yield.

3. Purify DNA by Centrifuge

1. Place a Quick Gel Extraction Column into a Wash Tube.
2. Load the dissolved gel solution onto the column (≤400 mg equivalent per column).
3. Centrifuge at >10,000 × g for 1 min. Discard flow-through.
4. Add 500 µL Wash Buffer (with ethanol) to the column.
5. Centrifuge again for 1 min; discard flow-through.
6. Centrifuge at max speed (1–2 min) to remove residual buffer.
7. Transfer the column to a clean Elution Tube.

4. Elute DNA

1. Add 50 µL Elution Buffer (E1) to the center of the column membrane.
2. Incubate 1 min at room temperature.
3. Centrifuge at >10,000 × g for 1 min.
4. Collect purified DNA (~48 µL recovery).

---

Notes and Tips

• Use a clean razor blade and sterile tubes to prevent DNase contamination.
• Larger fragments (>10 kb) may yield less efficiently.
• If yield is low, check for incomplete gel dissolution or repeat elution with +30 µL buffer.
• Ensure complete drying before elution to avoid ethanol carryover.

---

PCR Protocol

Materials Required

• DNA template (1 ng/µL; use 1 µL per reaction)
• Forward primer (10 µM)
• Reverse primer (10 µM)
• dNTP mix (10 mM each)
• 5× PCR buffer
• DNA polymerase (1 U/µL)
• Nuclease-free water
• PCR tubes or plates

Equipment

• Thermal cycler
• Micropipettes with sterile, filtered tips
• Ice bucket for reagent handling

---

Step 1. Prepare the Master Mix

Component	Stock Concentration	Volume (µL)	Final Concentration
5× PCR buffer	5×	10.0	1×
dNTP mix	10 mM each	1.0	200 µM each
Forward primer	10 µM	2.5	0.5 µM
Reverse primer	10 µM	2.5	0.5 µM
DNA template	1 ng/µL	1.0	1 ng total
DNA polymerase	1 U/µL	1.0	1 U
Subtotal		18.0	—
Nuclease-free water	—	32.0	—
Total	—	50.0	—

Preparation Steps

1. On ice, prepare a master mix (exclude template DNA).
2. Aliquot 49 µL of mix per PCR tube.
3. Add 1 µL template DNA, mix gently, then spin down.

---

Step 2. PCR Cycling Conditions

1. Initial Denaturation: 98 °C, 30 s
2. Amplification (30 cycles):
   • Denaturation: 98 °C, 10 s
   • Annealing: 30 s (temperature depends on primer Tm)
   • Extension: 72 °C, 15 s per kb
3. Final Extension: 72 °C, 2 min
4. Hold: 4 °C indefinitely

---

Notes and Tips

• Keep reagents on ice before loading.
• Include a no-template control (NTC) for contamination check.
• Optimize annealing temperature, Mg²⁺, or cycle number for low yields.

---

PCR Product Purification Using PureLink™ PCR Purification Kit

This protocol purifies PCR products by removing primers, dNTPs, enzymes, and salts.

Materials Supplied in the Kit

• Binding Buffer (B2)
• Binding Buffer High-Cutoff (HC/B3)
• Wash Buffer (W1)
• Elution Buffer (E1; 10 mM Tris-HCl, pH 8.5)
• PureLink™ Spin Columns and Collection Tubes
• Elution Tubes (1.7 mL)

Materials You Need to Provide

• 100% isopropanol
• 96–100% ethanol
• Microcentrifuge (≥10,000 × g)
• DNase-free pipettes and tips
• (Optional) Sterile distilled water (pH 7–8.5)

---

Buffer Preparation (First Use Only)

1. Add ethanol to Wash Buffer (W1):
   • 64 mL (for 16 mL bottle)
   • 320 mL (for 80 mL bottle)
2. Add isopropanol to:
   • Binding Buffer (B2): 10 mL (15 mL bottle) or 48 mL (72 mL bottle).
   • Binding Buffer HC (B3): 2.3 mL (23 mL bottle) or 11 mL (109 mL bottle).

---

Step-by-Step Protocol

1. Bind DNA

1. Mix 1 volume PCR product (50–100 µL) with 4 volumes Binding Buffer (B2 or B3).
   • Use B2 for 100 bp–12 kb fragments.
   • Use B3 to remove <300 bp fragments (e.g., primer dimers).
2. Load mixture into column; centrifuge at 10,000 × g for 1 min.
3. Discard flow-through.

2. Wash DNA

1. Add 650 µL Wash Buffer (with ethanol) to the column.
2. Centrifuge at 10,000 × g for 1 min.
3. Discard flow-through.
4. Centrifuge again at max speed (2–3 min) to dry.

3. Elute DNA

1. Place column in Elution Tube.
2. Add 50 µL Elution Buffer (E1) or sterile water (pH 7–8.5).
3. Incubate 1 min at room temperature.
4. Centrifuge at max speed (2 min).
5. Collect ~48 µL purified PCR product.
6. Store at –20 °C or use immediately.

---

Notes and Tips

• Pipette elution buffer directly to membrane center.
• Ensure ethanol/isopropanol added correctly before use.
• For maximum primer removal, use Binding Buffer HC (B3).

---

Recombinase Polymerase Amplification (RPA) Protocols — 2025

Objective

To verify that RPA amplification functions with EGFR mutation and wildtype templates.

---

Materials

• Thermo Fisher Lyo-ready RPA Kit Components:
  • Lyo-ready T4 UvsX Protein (1.2 mg/mL), 50 µL
  • Lyo-ready T4 UvsY Protein (1.2 mg/mL), 50 µL
  • Lyo-ready Bst DNA Polymerase (6 U/µL), 50 µL
  • Lyo-ready T4 Gene 32 Protein (16 mg/mL), 50 µL
  • 2× RPA Reaction Buffer, 1000 µL
  • 280 mM MgCl₂, 100 µL
  • 10 mM dNTP Mix, 50 µL
• Forward and reverse primers (starting at 0.3 µM each)
• Template DNA: mutant control, wildtype control, NTC (water)
• Nuclease-free water
• 0.2 mL PCR tubes
• Heat block or thermocycler with open heated lid
• Agarose gel, buffer, stain, and ladder for visualization

---

Reaction Setup (20 µL total)

Prepare on ice. Thaw, vortex, and briefly spin down all reagents.

Master Mix (per reaction)

1. 2× RPA Reaction Buffer: 10.0 µL
2. dNTP Mix (10 mM each): 0.4 µL
3. Forward Primer (10 µM): 0.6 µL
4. Reverse Primer (10 µM): 0.6 µL
5. Lyo-ready T4 UvsX Protein: 0.5 µL
6. Lyo-ready T4 UvsY Protein: 0.5 µL
7. Lyo-ready T4 Gene 32 Protein: 0.5 µL
8. Lyo-ready Bst DNA Polymerase: 0.5 µL
9. Nuclease-free water: to 19 µL
10. Template DNA: 1 µL (added individually)
11. MgCl₂ (280 mM): 1 µL (final 14 mM) — added last to start reaction.

---

Reaction Initiation

1. Dispense 19 µL master mix into each tube.
2. Add 1 µL template DNA (or water for NTC).
3. Add 1 µL MgCl₂, mix gently (pipette or flick), quick spin.

---

Incubation

• Incubate at 42 °C for 20 min.
• Mix briefly at 4–5 min and 9–10 min marks.
• For abundant templates, reduce to 10–15 min; for low copy number, extend to 25 min.

---

Post-Reaction Analysis

• Add loading dye.
• Run on 1–2% agarose gel with appropriate DNA ladder.
• Visualize expected amplicon size bands.

---