PCR Barcoding Protocol

Module 7: Magnetic Bead Extraction + PCR Setup Protocol

November 6, 2025 | 2:00-4:50 PM

The Ultimate Extraction Showdown

Protocol Overview

This protocol combines magnetic bead DNA extraction with COI PCR amplification. You will extract DNA from new mosquito samples using magnetic bead technology while simultaneously amplifying DNA from last week's column extractions. This represents real-world molecular lab workflows where multiple processes run in parallel.

Learning Outcomes

Master magnetic bead DNA extraction technology
Execute PCR setup with precious DNA samples
Compare two extraction methods quantitatively
Optimize PCR conditions for different DNA qualities
Integrate multi-tasking in molecular workflows

Assessment Criteria

Technical Excellence (35%): Bead handling, PCR setup precision
Time Management (25%): Running parallel processes efficiently
Data Analysis (25%): Method comparison, statistical thinking
Problem Solving (15%): Adapting to DNA quality differences

Today's Epic Quest

Your teams now extract DNA from new mosquitoes using magnetic beads. Can magnetic beads beat traditional columns? Plus, you'll amplify last week's DNA while extracting new samples. Welcome to real lab life!

Schedule (The Juggling Act)

2:00-2:15: Review previous module data, prepare stations
2:15-2:30: Start magnetic bead extractions (3 samples: -80°C, 95% ethanol, silica gel)
2:30-2:45: [Lysis incubation] Set up COI PCR with previously extracted column DNA samples
2:45-3:00: Start PCR program (2 hours)
3:00-3:30: Complete bead extractions
3:30-4:00: Qubit new samples from beads
4:00-4:15: Quick gel check of PCR (if time)
4:15-4:30: Data comparison and analysis
4:30-4:50: Document results

Part 1: Pre-Lab Strategy Session (15 min)

Review Previous Module Results

Which samples will amplify best?

Team Decision Tree

If DNA < 10 ng/µL → Use 5 µL template
If DNA 10-30 ng/µL → Use 2 µL template
If DNA > 30 ng/µL → Use 1 µL template

Today's Mosquitoes

Different batch from previous module
Species unknown
Same 3 preservation methods
Prediction time!

Part 2: BioDynami HMW Magnetic Bead Protocol (75 min)

The Gentle Giant Approach

Unlike violent centrifugation, magnetic beads whisper DNA away from contaminants!

Your 3 New Samples

Sample	Preservation	Current Yield (Beads)	Predicted Bead Advantage
D	-80°C frozen	___ ng/µL	× 1.5 = ___
E	95% ethanol	___ ng/µL	× 1.5 = ___
F	Silica gel	___ ng/µL	× 1.5 = ___

Step 1: Lysis (30 min)

1 Label tubes D-F clearly

2 Add mosquito to each tube

3 Add 200 µL Lysis Buffer

4 Grind gently (beads are sensitive!)

5 Add 20 µL Proteinase K

6 Mix by inversion (no vortex!)

7 Incubate 56°C for 30 min

Critical Step

NOW → Set up PCR (see Part 3)

While samples are lysing, you have time to set up PCR reactions with last week's DNA!

Step 2: Bead Binding (15 min)

8 Add 20 µL magnetic beads (mix first!)

9 Add 200 µL Binding Buffer

10 Mix by pipetting 10 times

11 Incubate 5 min room temp

12 Place on magnetic rack

13 Wait 2 min for separation

14 Remove supernatant carefully

Step 3: Washing (20 min)

15 Add 500 µL Wash Buffer 1

16 Remove from magnet

17 Mix by inversion

18 Back on magnet 2 min

19 Remove supernatant

20 Repeat with Wash Buffer 2

21 Air dry 5 min (not too long!)

Step 4: Elution (10 min)

22 Add 50 µL Elution Buffer

23 Remove from magnet

24 Mix by pipetting

25 Incubate 2 min

26 On magnet 2 min

27 Transfer clear supernatant

28 Store on ice

Part 3: PCR Setup (During Lysis) - 15 min

Mission: Amplify COI from Previously Extracted DNA

Master Mix (Make 10% extra for 6 reactions)

Component	1 Reaction (25 µL)	6 Reactions (×1.1)	Final Concentration
Q5® 2X Master Mix	12.5 µL	82.5 µL	1×
AU-COI Forward Primer (10 µM)	1.25 µL	8.25 µL	0.5 µM
AU-COI Reverse Primer (10 µM)	1.25 µL	8.25 µL	0.5 µM
Water (nuclease-free)	8.0 µL	52.8 µL	—
Total Master Mix	23 µL	151.8 µL	—

Q5® 2X Master Mix Optimization Notes

Primer Concentration (0.5 µM): Standard for Q5 High-Fidelity; lower than some protocols to reduce non-specific amplification while maintaining AU-COI primer specificity.

Master Mix Pre-formulation: Q5® 2X Master Mix (NEB #M0492) includes Q5 polymerase, dNTPs, and optimized buffer. Do NOT add separate dNTPs or buffer.

Template DNA Amount: Add 1-2 µL extracted DNA (aim for 10-50 ng total DNA per reaction) after master mix distribution.

Reaction Setup

1 Aliquot 23 µL master mix per tube

2 Add templates:

Tubes 1-3: 1-2 µL student DNA (previously extracted samples)
Tube 4: 1-2 µL known arthropod DNA (positive control)
Tube 5: 2 µL water (negative control)

3 Add water if needed to reach 25 µL total (0-1 µL)

4 Label clearly: Sample ID + (+) or (-)

5 Mix by pipetting

6 Quick spin

7 Load thermocycler

PCR Program - Standard COI Protocol (Approximately 2.5 hours)

94°C - 2 minutes (Initial denaturation)

Main Cycling Phase (35 cycles):
┌─ 94°C - 30 seconds (Denaturation)
├─ 48°C - 30 seconds (Annealing - Standard for COI Folmer/AU-COI)
└─ 72°C - 1 minute (Extension - 712 bp product)

72°C - 7 minutes (Final extension)
4°C - Hold (indefinitely)

NOTES:
- 94°C denaturation: Standard for COI gene amplification
- 48°C annealing: Standard for COI Folmer/AU-COI primers (LCO1490/HCO2198)
- 1 minute extension: Standard timing for ~710 bp amplicon
- 7 min final extension: Ensures complete product synthesis and polishing for Sanger sequencing
- 35 cycles: Optimal for mitochondrial COI amplification with high copy number

Part 4: Qubit Round 2 (30 min)

The Moment of Truth

Do beads beat columns?

Protocol (Same as Previous Module)

2 µL DNA + 198 µL working solution
Compare immediately to previous column results

Live Scoreboard

TEAM 1             TEAM 2
Columns    Beads     Columns    Beads
━━━━━━    ━━━━━     ━━━━━━    ━━━━━
-80°C: ___ vs ___   -80°C: ___ vs ___
95%:   ___ vs ___   95%:   ___ vs ___
Sil:   ___ vs ___   Sil:   ___ vs ___

Part 5: Quick Gel Visualization (15 min)

PCR Success Check

1 Load 5 µL PCR product + 1 µL dye

2 Run 100V for 10 min

3 Expected band: ~710 bp

4 Which preservation method amplified best?

Troubleshooting Guide

No band? Too little/degraded DNA
Smear? Too much DNA or degraded
Multiple bands? Non-specific amplification
Primer dimers? No template DNA

Control Interpretation

Positive control: Should show strong ~710 bp band
Negative control: Should be completely blank (no bands)
If negative control has bands → Contamination problem!
If positive control fails → PCR setup problem

Data Analysis & Comparison (15 min)

The Extraction Method Battle

Method	Pros	Cons	Best For
Columns	Fast, reliable	Harsh, losses	Standard samples
Beads	Gentle, HMW	Slower, expensive	Precious samples

Statistical Thinking

Calculate average improvement between weeks
Which method shows less variation?
Cost per sample analysis
Time per sample comparison

Real-Time Evaluation

Skill Checkpoints

2:30 - PCR set up during lysis? (Multitasking!)
3:00 - Beads fully separated? (Patience!)
3:30 - All samples extracted? (Efficiency!)
4:00 - PCR bands visible? (Success!)

Critical Thinking Questions

Why do beads sometimes give higher yields?
What's the molecular weight difference?
Which method for 1000 samples? For 1 precious sample?

Peer Review

Rate your partner's:

Bead handling technique: ___/5
PCR setup accuracy: ___/5
Data recording: ___/5

Legendary Achievements

Today's Badges

Speed Demon: Finished both procedures on time
Magnet Master: No bead loss during washing
PCR Pro: All samples amplified
Data Detective: Found surprising pattern
Prediction Prophet: Correctly predicted yields

Hall of Fame Entry

If your silica sample amplified successfully, you're excellent!

Post-Lab Assignment

Lab Report Section (Due Later)

Write methods section for your lab report (300 words):

Compare both extraction methods
Include actual yields (with statistics)
Justify PCR template amounts
Explain any failures

Preparation for Next Module

Research Sanger sequencing
Review gel electrophoresis interpretation
Read about PCR product cleanup

Exit Interview

Team Debrief

Which extraction method wins? Why?
Biggest surprise today?
What would you change?
Ready for sequencing?

Instructor Feedback

Technical skill level: ___/10
Problem-solving: ___/10
Team collaboration: ___/10
Areas to improve: __________

Next Module Preview

Module 8: Gel Electrophoresis & PCR Analysis

Run complete gel analysis of PCR products
Quantify amplification success
Clean PCR products for sequencing
Submit to UCR Core Facility
Learn about Sanger sequencing technology

The Ultimate Question

Did you have Wolbachia-positive mosquitoes all along?

Inspiration

"Today you didn't just extract DNA - you mastered two technologies and generated real data!"

"Remember: In molecular biology, timing is everything. PCR waits for no one!"