Feb 06, 2025
Specific (first) 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. Specific (first) PCR of the Leeselab 2-step PCR protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1dmy7vr2/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 29, 2025
Last Modified: February 06, 2025
Protocol Integer ID: 119269
Keywords: pcr, first PCR step, two-step PCR, pcr of the leeselab, first pcr step, product in the second pcr, step pcr protocol, step pcr protocol this protocol, specific first step of the leeselab, specific primer, step primer, second pcr, primer, pcr, universal primer, addition to the specific primer, well pcr plate, leeselab, specific first step, fwhr2n, target sequence, second step, bp tag
Abstract
This protocol describes how to perform the specific first step of the Leeselab 2-step PCR protocol in 10 µL reactions. In the first PCR step, universal primers (e.g. fwhF2/fwhR2n) are used toamplify the target sequence. There are up to 24 different first-step primers individually labeled with a 4 - 6 bp tag (see table in the material section). These are used to individually tag up to 24 different 96-Well PCR Plates. In addition to the specific primer and the tag, each of the primers contains a universal overhang, which is used to further amplify the product in the second PCR. In the second step of the 2-step-PCR, each well is tagged with an individual Index (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 Primer (universal), see Excel spreadsheet below
Reverse Primer (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 full-skirted PCR plates
Documents:
First_step_primer_Leeselab.xlsx9KB Protocol materials
Hard-Shell 96-well PCR plateBio-Rad LaboratoriesCatalog #HSP9601
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
PCR foil, DNase-/RNase-freeSarstedtCatalog #95.1993
Invitrogen UltraPure DNase/RNase-Free Distilled WaterFisher ScientificCatalog #11538646
Troubleshooting
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.
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
26m
Calculate the required volumes for the PCR Mastermix using the Excel spreadsheet.
Note
You can download the Exel spreadsheet here:
Mastermix_calculation.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). Samples 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, "1st_pcr", "A").
Multiplex Mastermix (M), forward (fwd) and reverse (rev) primer, PCR-grade water and PCR Mastermix (MM).
2m
Briefly vortex and shortly spin down all samples (DNA extract ) 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 , forward primer, reverse primer, and UltraPure™ DNase/RNase-Free Distilled WaterThermo Fisher ScientificCatalog #10977023 .
Preparation of the PCR Mastermix (MM).
Note
This step has to be performed separately per plate when using the tagging scheme mentioned above since per plate an individual combination of primers is used per plate.
3m
Briefly vortex and shortly spin down the prepared PCR Mastermix.
1m
Distribute 9 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 1 µL DNA extract per well. This step has to be done with a Multichannel pipette to ensure correct assignment and minimize the contamination risk.
Always maintain the order of samples (here top to bottom, left to right) for pipette tips, source plate and target plate (see below).
Use 1 µL of each sample (DNA extract).
Fill the DNA extracts in the respective wells (same order on each plate).
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 PCR plate.
1m
Close the extraction 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
2h 2m
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 fwhF2/fwhR2n:
Step 1: 95°C for 5 minutes (initial denaturation)
Step 2: 95°C for 30 seconds (denaturation)
Step 3: 58°C for 90 seconds (annealing)
Step 4: 72°C for 30 seconds (elongation)
--- repeat for 20 cycles ---
Step 5: 68°C for 10 minutes (final elongation)
Note
For samples with low template amounts (e.g. eDNA from water), also 30 cycles of 1st step PCR can be used.
2h
Storage
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