Jun 17, 2026

Simplified Protocol for Preparing PCR‑Competent Bacterial Lysates (Gram‑Positive and Gram‑Negative) for Teaching Laboratories V.2

DOI
https://dx.doi.org/10.17504/protocols.io.e6nvwx617gmk/v2
  • 1Universidad de Puerto Rico, Rio Piedras
Cristina L Andujar-Sierra
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DOI: https://dx.doi.org/10.17504/protocols.io.e6nvwx617gmk/v2
Protocol Citation: Cristina L Andujar-Sierra, abigail.strubbe , Yilmaz Koru, Cid M Calderon, miguel.urdaneta , Norma RodriguezGomez, Ariana Rodríguez-Flores, Lizbeth Alvarado-Vargas, Jose Agosto-Rivera 2026. Simplified Protocol for Preparing PCR‑Competent Bacterial Lysates (Gram‑Positive and Gram‑Negative) for Teaching Laboratories. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwx617gmk/v2Version created by Cristina L Andujar-Sierra
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 12, 2026
Last Modified: June 17, 2026
Protocol  Integer ID: 319068
Keywords: bacterial lysis, crude DNA extraction, Y-PER, thermal lysis, PCR template, 16S rRNA, colony lysis, teaching laboratory, amplification of the bacterial 16, use of conventional dna purification kit, conventional dna purification kit, bacterial 16, yeast protein extraction reagent, bacterial lysate, taq polymerase, inhibition of taq polymerase, rrna gene, residual nuclease, laboratories this protocol, amplification by agarose gel electrophoresi, commercial dna extraction kit, need for commercial dna extraction kit, agarose gel electrophoresi, 16s rrna gene with universal primer, generating pcr, negative bacteria, taxonomic identification by blast, teaching laboratory, confirming amplification, agarose gel, teaching laboratory environment
Funders Acknowledgements:
NSF Arecibo Center for STEM Education, Computing, and Community Engagement
Grant ID: 2321760
NSF Arecibo Center for STEM Education, Computing, and Community Engagement
Grant ID: 2321761
NSF Arecibo Center for STEM Education, Computing, and Community Engagement
Grant ID: 2321759
USDA-OPPE (NIFA)
Grant ID: AO242501X443G006
Abstract
This protocol describes a classroom-adapted workflow for generating PCR-competent bacterial lysates from single colonies using Y-PER chemical and thermal lysis, amplifying the 16S rRNA gene with universal primers 27F and 1492R, and confirming amplification by agarose gel electrophoresis without the need for commercial DNA extraction kits. The method is compatible with both Gram-positive and Gram-negative bacteria and is designed for teaching laboratory environments where time and resource constraints limit conventional extraction workflows. The expected result is a single ~1,450 bp amplification band on a 1.3% agarose gel stained with SYBR Safe, suitable for downstream Sanger sequencing and taxonomic identification by BLAST.
Guidelines
This protocol is designed for teaching laboratory environments. Instructor preparation is required before students arrive, see the Instructor Protocol document for pre-aliquoting instructions, master mix preparation, buffer preparation, and DNA ladder preparation. Students work with pre-labeled, pre-aliquoted tubes and do not prepare reagents themselves. One or two bacterial culture per student is required. The complete workflow (lysis + PCR setup) takes approximately 1.5 hours; PCR cycling runs for ~1 h 50 min. Gel electrophoresis is completed in a subsequent session.
Materials
Materials and Reagents

Cell Lysis: Reagents

  1. Reagent: Y-PER Yeast Protein Extraction Reagent
  • Supplier / Catalog: Thermo Scientific, cat. #78990
  • Storage: Room temperature
2. Reagent: Nuclease-Free Water (DNase/RNase-free)
  • Supplier / Catalog: Any supplier (confirm DNase/RNase-free)
  • Storage: Room temperature
3. Reagent: Bacterial colonies on solid medium
  • Supplier / Catalog: Grown on TSA or equivalent, 24–72 h, 37 °C; isolated colonies required
  • Storage: Use fresh

PCR: Reagents'
  1. Reagent: OneTaq 2X Master Mix with Standard Buffer*
  • Supplier / Catalog: New England Biolabs, cat. #M0482
  • Storage: −20 °C
2. Reagent: Primer 27F: 5'-AGAGTTTGATCMTGGCTCAG-3'
  • Supplier / Catalog: Integrated DNA Technologies (IDT), custom oligo order — working conc: 10 µM in nuclease-free water
  • Storage: −20 °C
3. Reagent: Primer 1492R: 5'-TACGGYTACCTTGTTACGACTT-3'
  • Supplier / Catalog: Integrated DNA Technologies (IDT), custom oligo order — working conc: 10 µM in nuclease-free water
  • Storage: −20 °C
4. Reagent: Nuclease-Free Water
  • Supplier / Catalog: Any supplier (DNase/RNase-free)
  • Storage: Room temperature

Gel Electrophoresis: Reagents
  1. Reagent: Agarose
  • Supplier / Catalog: Bio-Rad, cat. #161-3100
  • Storage: Room temperature
2. Reagent: TAE Buffer 10X
  • Supplier / Catalog: Sigma-Aldrich, cat. 
  • Storage: Room temperature
3. Reagent: SYBR Safe DNA Gel Stain
  • Supplier / Catalog: Thermo Fisher, cat. #S33102
  • Storage: Room temperature, protect from light
4. Reagent: Gel Loading Dye, Purple (6X)
  • Supplier / Catalog: New England Biolabs, cat. #B7025S
  • Storage: −20 °C
5. Reagent: 100 bp DNA Ladder
  • Supplier / Catalog: New England Biolabs, cat. #N3231
  • Storage: −20 °C
6. Reagent: Distilled or deionized water
  • Supplier / Catalog: Any supplier
  • Storage: Room temperature

Materials
  • Micropipettes, 0.5–10 µL range — For colony picking and small-volume transfers
  • Micropipettes, 2–20 µL range — General use
  • Sterile aerosol-barrier micropipette tips — Required for all PCR steps
  • PCR microtubes, 0.2 mL thin-wall — For lysis, PCR setup, and sample loading
  • Microcentrifuge tubes, 0.5 mL — For centrifuge adapter assembly (lysis)
  • Microcentrifuge tubes, 1.5 mL — For adapter assembly and master mix prep
  • Thermal cycler (Bio-Rad DNA Engine Peltier Thermal Cycler, Model PTC-0200) — For lysis (95 °C hold) and PCR cycling
  • Microcentrifuge capable of 10,000 × g — For lysate clarification
  • Agarose gel electrophoresis system (tank, tray, 8- to 12-well comb)
  • Power supply, 100 V capable — For electrophoresis run
  • Blue-light gel imaging system (Bio-Rad GelDoc Go, cat. #12009077) — For gel imaging
  • Erlenmeyer flask, 125–250 mL — For agarose preparation
  • Microwave oven — For melting agarose
  • Thermometer — For monitoring agarose cooling temperature
  • Graduated cylinder — For buffer preparation
  • Ice block or wet ice — For PCR setup and sample storage
  • Nitrile gloves — PPE, required throughout
Safety warnings
CAUTION:
  1. Do not allow OneTaq Master Mix to reach room temperature.
2. Do NOT pierce or scrape the agar surface. Agar polysaccharides are PCR inhibitors and will cause amplification failure (Gibb et al., 1998).
3. Pipette gently and slowly. Y-PER produces bubbles when agitated vigorously, which can interfere with accurate volume transfer. If bubbles form, briefly centrifuge to resolve.
4. Ensure all tubes are fully closed before starting the lysis program.
5. Do NOT touch the pellet. If disturbed, re-centrifuge for 2 additional minutes at 10,000 × g before transferring.
6. Change pipette tips between ALL samples and controls. Cross-contamination is the most common source of false positives in PCR.
7. The flask will be very hot. Use heat-resistant gloves or a towel at all times. Do not allow the solution to boil over.
8. Do NOT add SYBR Safe above 65 °C (stain degrades) or below 55 °C (gel may solidify before pouring).
9. Do not release the pipette plunger until the tip is fully withdrawn from the well. Releasing inside the well creates back-suction that pulls the sample out.
Before start
Before beginning, confirm that all instructor preparations are complete: bacterial colonies on TSA plates are isolated and fresh (18–24 h at 37 °C); student workstation tubes are pre-labeled and pre-aliquoted (Tube A: 20 µL Y-PER; Tube B: 180 µL nuclease-free water; Tube C: 18 µL PCR Master Mix on ice); thermal cycler is programmed with both the lysis (95 °C / 10 min) and PCR programs; and ice is available at every workstation. All reagents must remain on ice from this point forward. For complete instructor preparation instructions, see Steps 1–6.
Instructor Preparation: Complete Before Students Arrive
Verify Bacterial Colony Condition
Confirm that colonies are isolated (not confluent), grown on TSA or equivalent solid medium for 18–24 h at 37 °C. Mixed or confluent growth will compromise results. Pre-label two 0.2 mL PCR tubes per sample for lysis and one per sample for PCR before students arrive.
Thaw PCR Reagents
Remove OneTaq 2X Master Mix, primers 27F and 1492R, and nuclease-free water from -20 °C. Thaw fully on ice before use. Prepare ice block or wet ice and keep it available throughout the session.
CAUTION: Do not allow OneTaq Master Mix to reach room temperature.
Primer Dilution
Primers 27F and 1492R are received at a stock concentration of 100 µM. Dilute each primer to a working concentration of 10 µM using a 1:10 dilution before preparing the master mix.
  • Transfer 1 µL of primer stock (100 µM) into a sterile 1.5 mL microcentrifuge tube.
  • Add 9 µL of nuclease-free water to the same tube.
  • Mix thoroughly by pipetting up and down or by brief vortex. Final volume: 10 µL at 10 µM.
  • Repeat for the second primer.
Note: Label each tube with the primer name (27F or 1492R), working concentration (10 µM), and date of preparation. Store at -20 °C until use.
Prepare PCR Master Mix
Prepare the master mix in a 1.5 mL microcentrifuge tube on ice. Combine in the following order:

ComponentVolume per reactionConcentration
Nuclease-Free Water7.2 µL—
OneTaq 2X Master Mix10.0 µL1X
Primer 27F (10 µM)0.4 µL0.2 µM
Primer 1492R (10 µM)0.4 µL0.2 µM
Total pre-template volume18.0 µL—
Note: For multiple reactions, scale with 5–10% volume excess. Example for 18 reactions (5% excess): Water 136.1 µL · OneTaq 189.0 µL · 27F 7.56 µL · 1492R 7.56 µL. Mix by gentle inversion do NOT vortex. Once prepared, aliquot 18 µL into each pre-labeled student tube (Tube C) and keep on ice until the session begins.

Prepare Running Buffer (TAE 1X)
Prepare 1X TAE buffer fresh on the day of the lab session. To prepare 1 L of 1X working buffer from 10X stock (C₁V₁ = C₂V₂):
ComponentVolume
TAE Buffer 10X (Sigma-Aldrich, cat. #574797)100 mL
Distilled or deionized water900 mL
Total1,000 mL (1X)
Note: Label the bottle with buffer name, concentration (1X), and preparation date. TAE is preferred when downstream applications such as Sanger sequencing are planned (Obrador-Sánchez et al., 2017).

Prepare DNA Ladder
Prepare one ladder tube per gel in a 0.2 mL PCR tube on ice:
ComponentVolume
100 bp DNA Ladder (NEB, cat. #N3231)1 µL
Gel Loading Dye 6X (NEB, cat. #B7025S)1 µL
Distilled water4 µL
Total volume6 µL
Note: Keep the ladder on ice until loading. If running 4 gels, prepare 4 ladder tubes one per gel.
Procedure: Chemical and Thermal Cell Lysate
10m
Confirm that a pre-labeled 0.2 mL PCR tube containing 20 µL of Y-PER is already at your workstation before you begin.
Note: Tubes have been pre-aliquoted by the instructor. Do not transfer to a different tube.
Using a sterile micropipette tip (0.5–10 µL range) or a disposable inoculation loop, gently touch the surface of a single isolated bacterial colony. Transfer the tip or loop directly into the 20 µL of Y-PER in your pre-labeled tube and swirl gently to release the bacterial material.
CAUTION: Do NOT pierce or scrape the agar surface. Agar polysaccharides are PCR inhibitors and will cause amplification failure (Gibb et al., 1998).
Resuspend the bacterial colony by pipetting up and down gently with the same tip, or by swirling the loop, until the suspension appears uniformly turbid.
Note: Target turbidity: visibly cloudy but not opaque. If the suspension is clear, retrieve an additional colony with the same tip or a new loop and repeat. If very dense, plan to dilute the final lysate further before PCR.

Figure 1. Colony picking and resuspension technique. (A) Picking cells from a colony. (B) Cells on a sterile tip. (C)Suspending cells in Y-PER. (D) Cell suspension turbidity reference: 1 = water only; 2 = correct turbidity; 3 = too few cells; 4 = too many cells.

CAUTION: Pipette gently and slowly. Y-PER produces bubbles when agitated vigorously, which can interfere with accurate volume transfer. If bubbles form, briefly centrifuge to resolve.
Thermal Lysis: Cap the tube. Place in the thermocycler and run the lysis program: 95 °C for 10 minutes, then hold at 4 °C .
Note: This thermal step completes disruption of cells not fully lysed by Y-PER by denaturing structural membrane proteins and increasing membrane permeability, thereby releasing residual intracellular contents (Islam et al., 2017).
CAUTION: Ensure all tubes are fully closed before starting the lysis program.
When the Thermal Lysis is finished, prepare the centrifuge with the adapters for the tubes. If a PCR tube centrifuge adapter is not available, assemble one as follows: remove the cap from a 1.5 mL microcentrifuge tube; insert a 0.5 mL tube inside it; then place the 0.2 mL PCR tube inside the 0.5 mL tube. Centrifuge at 10,000 × g for 5 minutes.
10m
Carefully take 19 µL of supernatant without disturbing the pellet. Transfer to a new tube containing 180 µL of nuclease-free water. Mix gently.
Note: This 1:10 dilution reduces the concentration of detergent and cellular debris to levels known to inhibit PCR less frequently  (Sidstedt et al., 2020). The pellet contains cell debris and is discarded. 
CAUTION: Do NOT touch the pellet. If disturbed, re-centrifuge for 2 additional minutes at 10,000 × g before transferring.
Diluted supernatant is the PCR-ready crude lysate. Use2 µL for PCR reaction.
Note: Crude lysates can be stored at 4 °C for short‑term use or at −20 °C for longer periods, consistent with general recommendations for detergent‑based lysates from (Packeiser et al., 2013)
Procedure: PCR Reaction Preparation
Confirm that you have one pre-labeled 0.2 mL PCR tube containing 18 µL of Master Mix on ice for each of your samples. If any tube is missing a label, label it now before continuing.
Note: The no-template control (NTC) tube is prepared and handled by the instructor. The NTC is: 18 µL of Master Mix with Sterile Nuclease free Water.
Add 2 µL of crude lysate to each of your sample tubes. The instructor will add 2 µL of nuclease-free water to the NTC tube.
CAUTION: Change pipette tips between ALL samples and controls. Cross-contamination is the most common source of false positives in PCR.
Cap all tubes securely. Briefly centrifuge with a 5–10 second pulse to deposit all liquid at the bottom of each tube.
Place tubes in the thermocycler. Run the following program:

Initial denaturation94 °C3 min1
Denaturation94 °C30 s 
Annealing55 °C30 s35
Extension68 °C90 s 
Final extension68 °C5 min1
Hold4 °CIndefinite∞
Note: Total run time: approximately 1 h 50 min for 35 cycles. 
 After the program is complete, proceed to gel electrophoresis, or store PCR products at 4 °C for short-term use (up to 48 h) or at −20 °C for long-term storage, consistent with general recommendations for DNA stability (New England Biolabs, 2024).
Procedure: Agarose Gel Electrophoresis
Weigh 0.52 g of agarose powder and place into a 125–250 mL Erlenmeyer flask.
Note: 0.52 g in 40 mL of buffer = 1.3% (w/v). Each gel tray uses 40 mL.
Add 30 mL of 1X buffer to the flask. Add the agarose. Then add the remaining 10 mL of buffer, rinsing the flask walls to recover any agarose residue. Swirl gently to distribute.
Heat in the microwave in intervals: 45 seconds, swirl; then 30-second intervals, swirling between each, until the solution is completely clear with no visible particles.
CAUTION: The flask will be very hot. Use heat-resistant gloves or a towel at all times. Do not allow the solution to boil over.
Allow to cool to approximately 60–65 °C. Use a thermometer to monitor temperature. This typically takes 10–15 minutes depending on room temperature.
Note: Do not leave the flask unattended during cooling; the agarose can solidify quickly once it drops below 55 °C, making it impossible to pour. Avoid placing the flask directly on a cold bench surface, as this causes the agarose to solidify from the bottom up unevenly before it can be poured into the tray. Hold the flask or rest it on a folded paper towel while monitoring temperature..
CAUTION: Add SYBR Safe only after the agarose has cooled to a comfortable handling temperature (according to manufacturer recommendations; Thermo Fisher Scientific, 2023), to prevent evaporation or premature gel solidification.
Add 4.5 µL of SYBR Safe DNA Gel Stain (1X final concentration). Mix gently by swirling; avoid creating bubbles.
Note: Protect SYBR Safe from prolonged light exposure at all times.
Place an 8 to12 well comb in the gel tray. Pour the agarose solution into the tray and allow to solidify completely at room temperature (approximately 20–30 minutes). Do not move the tray while solidifying
Once solidified, carefully remove the comb by pulling it straight upward without tearing the wells. The gel is now ready for loading.
Prepare the loading mix for each PCR sample in a 0.2 mL PCR tube on ice:
Mix gently by pipetting. Maintain on ice until loading.
PCR product5 µL
Gel Loading Dye 6X (NEB B7025S)1 µL
Total volume6 µL
Note: The loading dye serves two purposes: it adds density to the sample so it sinks into the gel well instead of diffusing into the buffer, and it contains visible tracking dyes that migrate with the DNA during electrophoresis, allowing you to monitor the progress of the run without stopping it. Without loading dye, samples would float out of the wells and the run could not be monitored visually.
Mix gently by pipetting. Maintain on ice until loading.
Verify that you have the pre-labeled DNA ladder tube at your workstation, prepared by your instructor. Keep it on ice until loading.
Place the solidified agarose gel in the electrophoresis tank. Add 1X buffer the same buffer used to prepare the gel until the gel is completely submerged.
Load 6 µL of the prepared DNA ladder into the first well. In the second well, load 6 µL of the NTC immediately after the ladder. (This step will be performed by the instructor.)
Load 6 µL of each prepared sample into the remaining wells. Position the pipette tip inside the well without piercing the gel; dispense slowly and steadily.
CAUTION: Do not release the pipette plunger until the tip is fully withdrawn from the well. Releasing inside the well creates back-suction that pulls the sample out.
Verify that the wells are oriented toward the negative electrode (black). DNA migrates toward the positive electrode (red).
Connect the power supply and run the gel at 100 V for 30–35 minutes, or until the loading dye front approaches the end of the gel.
Note: Monitor periodically. Do not allow the dye front to run off the gel.
After the run, the instructor will image the gel using the Bio-Rad GelDoc Go Imaging System and document the image
Note: Expected result: A single band of approximately 1,450 bp in all positive samples. The NTC must show NO band. Use the ladder image below to confirm band size  the expected PCR product migrates between the 1,500 bp and 1,200 bp bands of the 100 bp DNA Ladder (NEB).

Figure 2. 100 bp DNA Ladder (New England Biolabs). Size range: 100–1,517 bp. The 1,000 bp reference band appears brightest. The expected ~1,450 bp PCR product migrates between the 1,517 bp and 1,200 bp bands.

Protocol references
Church, D. L., Cerutti, L., Gürtler, A., Griener, T., Zelazny, A., & Emler, S. (2020). Performance and application of 16S rRNA gene cycle sequencing for routine identification of bacteria in the clinical microbiology laboratory. Clinical Microbiology Reviews, 33(4), e00053-19. https://doi.org/10.1128/CMR.00053-19

 Frank, J. A., Reich, C. I., Sharma, S., Weisbaum, J. S., Wilson, B. A., & Olsen, G. J. (2008). Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes. Applied and Environmental Microbiology, 74(8), 2461–2470. https://doi.org/10.1128/AEM.02272-07

Islam, M. S., Aryasomayajula, A., & Selvaganapathy, P. R. (2017). A review on macroscale and microscale cell lysis methods. Micromachines, 8(3), 83. https://doi.org/10.3390/mi8030083

 New England Biolabs. (2024). OneTaq 2X Master Mix with Standard Buffer (M0482). https://www.neb.com/products/m0482-onetaq-2x-master-mix-with-standard-buffer

New England Biolabs. (2024). 100 bp DNA Ladder. https://www.neb.com/products/n3231-100-bp-dna-ladder

 Packeiser, H., Lim, C., Balagurunathan, B., Wu, J., & Zhao, H. (2013). An extremely simple and effective colony PCR procedure for bacteria, yeasts, and microalgae. Applied Biochemistry and Biotechnology, 169(2), 695–700. https://doi.org/10.1007/s12010-012-0043-8

Thermo Scientific. (2023). Y-PER Yeast Protein Extraction Reagent user guide (MAN0011382). Thermo Fisher Scientific. https://assets.fishersci.com/TFS-Assets/LSG/manuals/MAN0011382_YPER_Yeast_Protein_Extract_Reag_UG.pdf

New England Biolabs. (2024). OneTaq 2X Master Mix with Standard Buffer (M0482). https://www.neb.com/products/m0482-onetaq-2x-master-mix-with-standard-buffer'

Sidstedt, M., Rådström, P., & Hedman, J. (2020). PCR inhibition in qPCR, dPCR and MPS — mechanisms and solutions. Analytical and Bioanalytical Chemistry, 412(9), 2009–2023. https://doi.org/10.1007/s00216-020-02490-2

Gibb, A. P., Tribuddharat, C., Moore, R. A., Louie, T. J., Levett, P. N., Laupland, K. B., & Church, D. L. (1998). Inhibition of PCR by agar and agarose. Journal of Clinical Microbiology, 36(1), 275–276. https://doi.org/10.1128/jcm.36.1.275-276.1998
Acknowledgements
This protocol was supported by the NSF Arecibo Center for STEM Education, Computing, and Community Engagement (Grant Nos. 2321760, 2321761, 2321759) and USDA-OPPE (NIFA) (Grant No. AO242501X443G006).