Time-tested protocols that work from the field to the lab
Rolling out now:
Enhanced access to the Kartzinel Lab's protocol repository
Check back often or contact the PI for additional info
Enhanced access to the Kartzinel Lab's protocol repository
Check back often or contact the PI for additional info
*New* protocols posted in ***October 2025***
Field-to-Lab
Dietary DNA Collections
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Diet Banking Protocol: Strategy to plan and implement a low-cost 'bank' of fecal samples suitable for use in studies of animal diets, microbiomes, and other nutritional metrics. In some cases these samples may also be useful for population genetic / genomic analyses. We customize this protocol to serve partners working under a wide variety of field conditions and with different levels of experience.
Dietary DNA Extraction Protocol: Our most popular and frequently used DNA extraction protocols are based on Zymo Quick-DNA Fecal/Soil Microbe Mini Prep Kits. This protocol includes discussion of how we choose between using the Zymo Standard Lysis Buffer vs. Zymo DNA/RNA Shield Buffer when planning projects. It details critical considerations and improvements that we have made through our projects over many years to ensure we can succeed under a huge variety of conditions. |
Lab video demonstrating how we prepare our bench to extract DNA from scat samples |
Plant Voucher & DNA Barcode Collections
Plant Voucher & DNA Collection Protocol: Exquisitely detailed protocol for collecting plant voucher specimens, preserving DNA barcode samples, and curating metadata. This version of our protocol is tailored to our most popular use-case: constructing an extensive and expert-verified plant DNA barcode library for a region.
Wildlife Parasite Voucher & DNA Barcode Collections
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Parasitic Helminth DNA Extraction: This protocol details our strategy for routine extraction of helminth parasite DNA. The protocol is based on Qiagen's Blood & Tissue Kit, but includes some helpful tips based on our experience overcoming challenges arising from (1) the variable size and quality of helminth specimens obtained from necropsy and (2) the difficulty digesting hard cuticles associated with certain taxa.
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Lab video demonstrating how we prepare helminth samples for DNA extraction
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PCR & Gel Electrophoresis
Plant DNA Barcoding
trnL c/d: Protocol to develop a reference library to which dietary trnL-P6 sequences may be matched (P6 is a short loop within trnL). It is also often used for intraspecific population genetic or phylogeographic studies requiring variation in chloroplast DNA haplotypes.
rbcLa: Protocol used to sequence rbcL, one of the 'standard' plant DNA barcode markers. This protocol was adapted from one previously used at the Smithsonian Institution.
matK: Protocol used to sequence matK, one of the 'standard' plant DNA barcode markers. This protocol was adapted from one previously used at the Smithsonian Institution and it represents our starting point for sequencing barcodes from a diversity of taxa at this marker.
trnH-psbA: Protocol used to sequence trnH-psbA, one of the 'standard' plant DNA barcode markers. This protocol was adapted from one previously used at the Smithsonian Institution and it represents our starting point for sequencing barcodes from a diversity of taxa at this marker.
Plant ITS-1: A versatile protocol for sequencing the first Internal Transcribed Spacer (ITS) in plants. It is a rewarding DNA barcode to use for a wide diversity of plants.
Plant ITS-2: Protocol for sequencing the second Internal Transcribed Spacer (ITS) in plants. This marker and primer pair have been recommended by Andreas Kolter and we have found them to be very useful as well.
rbcLa: Protocol used to sequence rbcL, one of the 'standard' plant DNA barcode markers. This protocol was adapted from one previously used at the Smithsonian Institution.
matK: Protocol used to sequence matK, one of the 'standard' plant DNA barcode markers. This protocol was adapted from one previously used at the Smithsonian Institution and it represents our starting point for sequencing barcodes from a diversity of taxa at this marker.
trnH-psbA: Protocol used to sequence trnH-psbA, one of the 'standard' plant DNA barcode markers. This protocol was adapted from one previously used at the Smithsonian Institution and it represents our starting point for sequencing barcodes from a diversity of taxa at this marker.
Plant ITS-1: A versatile protocol for sequencing the first Internal Transcribed Spacer (ITS) in plants. It is a rewarding DNA barcode to use for a wide diversity of plants.
Plant ITS-2: Protocol for sequencing the second Internal Transcribed Spacer (ITS) in plants. This marker and primer pair have been recommended by Andreas Kolter and we have found them to be very useful as well.
Mammal DNA Barcoding
Mitochondrial D-loop (mammals): Protocol to amplify the D-loop of the mitochondrial control region in diverse mammals. We have used this protocol to confirm the identity of mammalian hosts in fecal DNA metabarcoding / microbiome studies as well as to infer phylogenies. We have published versions of the protocol that use other polymerases (e.g., Amplitaq Gold), but this version uses standard NEB taq (cost effective for routine use).
Mitochondrial 16S (mammals): Protocol to amplify 16S in diverse mammals. We have used this protocol to confirm the identity of mammalian hosts in fecal DNA metabarcoding / microbiome studies as well as to infer phylogenies. This version uses= Amplitaq Gold, but could easily be converted to a standard NEB taq to improve costs for routine use.
Mitochondrial 16S (mammals): Protocol to amplify 16S in diverse mammals. We have used this protocol to confirm the identity of mammalian hosts in fecal DNA metabarcoding / microbiome studies as well as to infer phylogenies. This version uses= Amplitaq Gold, but could easily be converted to a standard NEB taq to improve costs for routine use.
Helminth DNA Barcoding
*New* Mitochondrial 16S (parasitic nematodes): Protocol to amplify mitochondrial 16S in parasitic nematodes from: Spirurida, Oxyurida, and Ascaridida ("Clade 3"); Rhabditida ("Clade 4"); Strongylida ("Clade 5"). The protocol us useful for both standard barcoding and DNA metabarcoding since the amplicon size is ~240 bp.
Herbivore Dietary DNA Metabarcoding
trnL-P6 g/h: This is the Kartzinel Lab’s standard PCR protocol for dietary DNA metabarcoding involving herbivores with the trnL-P6 marker. The primers have been modified to include Nextera-XT overhangs that permit Illumina library preps using Nextera protocols. This protocol eliminates artificial banding caused by the replication of Nextera overhangs compared to earlier protocols. We often consider the pros and cons of protocols to sequence this marker using primers for trnL-P6 g/h vs. trnL-P6 c/h based on project-specific research objectives, DNA barcode library coverage, and the sequencing platform we plan to use.
*New* trnL-P6 c/h: This is a more recent PCR protocol for dietary DNA metabarcoding involving herbivores with the trnL-P6 marker that we use in the Kartzinel Lab. This primer pair amplifies a larger fragment of the trnL gene than do primers g/h (which amplify a fully internal fragment to these). The advantage of a longer fragment is greater resolution, in principle, but in practice there are fewer publicly available reference sequences that span the full length of this marker. The primers have been modified to include Nextera-XT overhangs that permit Illumina library preps using Nextera protocols. This protocol eliminates artificial banding caused by the replication of Nextera overhangs compared to earlier protocols. We often consider the pros and cons of protocols to sequence this marker using primers for trnL-P6 g/h vs. trnL-P6 c/h based on project-specific research objectives, DNA barcode library coverage, and the sequencing platform we plan to use.
*New* trnL-P6 c/h: This is a more recent PCR protocol for dietary DNA metabarcoding involving herbivores with the trnL-P6 marker that we use in the Kartzinel Lab. This primer pair amplifies a larger fragment of the trnL gene than do primers g/h (which amplify a fully internal fragment to these). The advantage of a longer fragment is greater resolution, in principle, but in practice there are fewer publicly available reference sequences that span the full length of this marker. The primers have been modified to include Nextera-XT overhangs that permit Illumina library preps using Nextera protocols. This protocol eliminates artificial banding caused by the replication of Nextera overhangs compared to earlier protocols. We often consider the pros and cons of protocols to sequence this marker using primers for trnL-P6 g/h vs. trnL-P6 c/h based on project-specific research objectives, DNA barcode library coverage, and the sequencing platform we plan to use.
Gut Microbiomes
Bacterial 16S-V4: This is the Kartzinel Lab’s standard PCR protocol for microbiome analyses. It is based on a protocol by the Earth Microbiome Project using primers 515f and 806r. We have modified these primers to include Nextera-XT overhangs that permit Illumina library preps using Nextera protocols.
DNA Sequencing
Sanger Sequencing
*New* ExoSAP-IT (diluted): We most often use enzymatic clean-ups based on a cost-saving ExoSAP-IT protocol. We have had many years of success diluting the ExoSAP-IT reagent substantially to get more value from each tube we purchase. This document includes links to GoogleDrive files with access to personnel at Brown University related to institutional sequencing submission procedures. We hope the protocol itself is fundamentally useful for everyone who does this kind of sequencing and that the links only make the document especially valuable for our friends at Brown.
Gel DNA extraction [coming soon]
Gel DNA extraction [coming soon]
Amplicon sequencing: Oxford Nanopore MinION
- Coming soon!