Feb 06, 2025
Universal (second) PCR of the Leeselab 2-step PCR protocol
- Marie-Thérése Werner1,
- Dominik Buchner1
- 1University of Duisburg-Essen, Aquatic Ecosystem Research
- Aquatic Ecosystem Research
Protocol Citation: Marie-Thérése Werner, Dominik Buchner 2025. Universal (second) PCR of the Leeselab 2-step PCR protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzkq34vx1/v1
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: January 30, 2025
Last Modified: February 06, 2025
Protocol Integer ID: 119326
Keywords: pcr, second pcr step, two-step PCR
Abstract
This protocol describes how to perfom the universal second step of the Leeselab 2-step PCR protocol in 10 µL reactions. In the first PCR step, specific primers (e.g. fwhF2/fwhR2n) are used to tag each plate with an individual combination of tags (1-24) and a universal tail. In the second step of the 2-step-PCR, each well is tagged with an individual index sequence (1-96) enabling the identification of each sample (well) after library sequencing.
Guidelines
Follow general lab etiquette. Wear gloves to prevent contaminating the samples. Clean the workspace before starting with 80% EtOH.
Materials
Below all materials needed for the protocol are listed. Vendors and part numbers are listed but interchangeable.
Chemicals:
Multiplex Mastermix 5x Hot-Start Multiplex PCR-MastermixBio&SELLCatalog #BS91.522.1000MPP
Forward and reverse primers (universal), see Excel spreadsheet below
PCR-grade water Invitrogen UltraPure DNase/RNase-Free Distilled WaterFisher ScientificCatalog #11538646
Labware:
Micro-Twist-Top-Tube Micro-Twist-Top-TubeSarstedtCatalog #72.693
PCR plate Hard-Shell 96-well Hard-Shell 96-well PCR plateBio-Rad LaboratoriesCatalog #HSP9601
PCR foil, DNase-/RNase-free PCR foil, DNase-/RNase-freeSarstedtCatalog #95.1993
Devices:
Vortexer
Centrifuge for SBS format microplates
Table centrifuge for tubes
Pipettes and Multichannel pipettes including pipette tips
Thermocycler compatible wth low profile PCR plates
Documents:
Second_step_primer_Leeselab.xlsx16KB Protocol materials
Micro-Twist-Top-TubeSarstedtCatalog #72.693
5x Hot-Start Multiplex PCR-MastermixBio&SELLCatalog #BS91.522.1000MPP
UltraPure™ DNase/RNase-Free Distilled WaterThermo Fisher ScientificCatalog #10977023
Hard-Shell 96-well PCR plateBio-Rad LaboratoriesCatalog #HSP9601
Hard-Shell 96-well PCR plateBio-Rad LaboratoriesCatalog #HSP9601
Hard-Shell 96-well PCR plateBio-Rad LaboratoriesCatalog #HSP9601
Micro-Twist-Top-TubeSarstedtCatalog #72.693
Hard-Shell 96-well PCR plateBio-Rad LaboratoriesCatalog #HSP9601
Hard-Shell 96-well PCR plateBio-Rad LaboratoriesCatalog #HSP9601
Hard-Shell 96-well PCR plateBio-Rad LaboratoriesCatalog #HSP9601
PCR foil, DNase-/RNase-freeSarstedtCatalog #95.1993
Micro-Twist-Top-TubeSarstedtCatalog #72.693
Hard-Shell 96-well PCR plateBio-Rad LaboratoriesCatalog #HSP9601
PCR foil, DNase-/RNase-freeSarstedtCatalog #95.1993
5x Hot-Start Multiplex PCR-MastermixBio&SELLCatalog #BS91.522.1000MPP
Invitrogen UltraPure DNase/RNase-Free Distilled WaterFisher ScientificCatalog #11538646
Safety warnings
Reagents are potentially damaging to the environment. Dispose waste responsibly.
Before start
Make sure all materials are available before starting. Make sure all materials and samples are unfrozen.
This protocol uses clean first step PCR product as input material, e.g. prepared according to the following protocol:
Protocol
NAME
PCR cleanup and size selection with magnetic beadsCREATED BY
Dominik Buchner
Note
The protocol described here is designed for the use of PCR plates, but can also be done in tubes, strips, or any sufficient reaction vessel. The recommended shaking speeds are adjusted to the plates mentioned in the materials.
Preparations
Preparations
30m
30m
Calculate the required volumes for the PCR Mastermix using the Excel spreadsheet.
Note
You can download the Exel spreadsheet here:
Mastermix_calculation_2.xlsx 1m
Prepare the workplace to ensure contamination-free handling.
Workspace prepared for a right-handed person to work from left (pipette tips, samples) to right (waste). Clean PCR products and (unused) labware are placed in the back.
2m
Label the Micro-Twist-Top-TubeSarstedtCatalog #72.693 (e.g., "MM") and Hard-Shell 96-well PCR plateBio-Rad LaboratoriesCatalog #HSP9601 (e.g. project name, date, "2nd_pcr", "A").
2m
Briefly vortex and shortly spin down the clean PCR products and required chemicals.
2m
Pipette the PCR Mastermix in a Micro-Twist-Top-TubeSarstedtCatalog #72.693 containing the calculated volumes of 5x Hot-Start Multiplex PCR-MastermixBio&SELLCatalog #BS91.522.1000MPP and UltraPure™ DNase/RNase-Free Distilled WaterThermo Fisher ScientificCatalog #10977023 .
Note
Do not include the tagging primers in the master mix. Each well has to be tagged separately.
3m
Briefly vortex and shortly spin down the prepared PCR Mastermix.
1m
Distribute 6 µL per sample in each well used of the Hard-Shell 96-well PCR plateBio-Rad LaboratoriesCatalog #HSP9601 . To speed up this step, a Multidispenser can be used.
Distribution of the PCR Mastermix (MM) in all required wells.
8m
Pipette 2 µL primer mix [100 µM] per well.
Note
To ensure correct assignment and minimize the contamination risk, it is best to prepare a tagging primer plate and add the primer mix with a Multichannel pipette.
4m
Pipette 2 µL clean PCR product per well. This step has to be done with a Multichannel pipette to ensure correct assignment and minimize the contamination risk.
Note
Always maintain the order of samples (e.g., top to bottom, left to right) for pipette tips, source plate and target plate (pictures available in protocol below).
Protocol
NAME
Specific (first) PCR of the Leeselab 2-step PCR protocol
CREATED BY
Marie-Thérése Werner
4m
Close the Hard-Shell 96-well PCR plateBio-Rad LaboratoriesCatalog #HSP9601 with PCR foil, DNase-/RNase-freeSarstedtCatalog #95.1993 . Make sure that each well is properly sealed.
Sealed clean PCR products plate (back) and 2nd PCR plate with foil (front).
1m
Close the clean PCR product plate with PCR foil. Make sure each well is properly sealed.
1m
Briefly vortex and shortly spin down the sealed Hard-Shell 96-well PCR plateBio-Rad LaboratoriesCatalog #HSP9601 .
1m
PCR
PCR
1h 32m
1h 32m
Place the sealed Hard-Shell 96-well PCR plateBio-Rad LaboratoriesCatalog #HSP9601 in a Thermocycler and close it properly.
1m
Depending on the Thermocycler, a lid or pad should be placed on the PCR plate before closing the Thermocycler.
1m
Enter/select the correct program (depending on used primers and chemicals) and start the polymerase chain reaction.
Note
PCR program for universal Illumina primers:
Step 1: 95°C for 5 minutes (initial denaturation)
Step 2: 95°C for 30 seconds (denaturation)
Step 3: 61°C for 90 seconds (annealing)
Step 4: 72°C for 30 seconds (elongation)
--- repeat for 25 cycles ---
Step 5: 68°C for 10 minutes (final elongation)
1h 30m
Storage
Storage
1m
1m
Vortex and spin down the Hard-Shell 96-well PCR plateBio-Rad LaboratoriesCatalog #HSP9601 to use it for further processing, e.g. gel electrophoresis, and/or store it at 4°C (short-term) or -20°C (long term).
1m