Kartzinel:Laboratory Protocols

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All lab users must complete training requirements and consult with PI Tyler Kartzinel before beginning research in the lab. This wiki is also required reading. Please refer back to it regularly because policies and protocols are subject to change as we improve our work. If details are missing, do not assume they are unwanted; consult with Tyler to identify appropriate protocols and remember to report successes for inclusion on the wiki.

Pre- and Post-PCR

The lab will adhere to strict pre- and post-PCR policies. The general flow of materials through the DNA lab will be sample organization and extraction (pre-PCR freezer room and Biosafety cabinet), PCR setup (PCR hood), and post-PCR processes (thermocycler, gel rig, post-PCR freezer).

  • Samples, extract, and pre-PCR reagents must be kept in the pre-PCR room.
  • PCR reactions will be set up in the pre-PCR room, then moved to the thermocycler in the post-PCR room. All post-PCR applications must happen in the post-PCR room.
  • There is a designated set of sample racks for sample organization and extraction (green or blue), PCR setup (pink or purple), transfer (orange), and post-PCR applications (yellow).
  • Samples, reagents, and materials that need to move from the pre-PCR room to the post-PCR room have to happen in transfer containers. These are racks, coolers, and tubs that will NEVER BE USED WITH OPEN SAMPLES in either room. The sole purpose of the transfer vehicles is to physically move your stuff from one room to the other. They should never be used around pre-PCR samples or reagents (to avoid accidentally introducing post-PCR products) and they should never be used around post-PCR samples or reagents (to avoid accidentally picking up post-PCR products). Transfer containers are available for PCR strip tubes/plates, Qubit assay tubes, milliQ water, etc.
  • The transfer station in the pre-PCR room is designated next to the emergency eyewash station, to avoid accumulating post-PCR aerosols. Ensure the transfer racks are stored in their container. Keep container closed. Surface-sterilize transfer racks with bleach immediately upon returning them to the pre-PCR room.
  • The transfer station in the post-PCR room is designated on the metal rack near the door.

General Guidelines

Cleaning lab resources

  • All lab users are responsible for the cleaning and maintenance of each piece of lab equipment each time they use it (see equipment-specific details below).
  • All lab users are responsible for cleaning and storing the shared tube racks and trays that they used. If cleaning requires soaking something, please do not forget to return and finish the clean up as soon as appropriate.

Cleaning solutions

We often make bleach and ethanol solutions in the lab. These are critical for biosafety. [Link to Making and using bleach solutions]

Freezers

Notes on using and organizing freezer materials. [Link to Freezers page]

Pipettes

Notes on using and caring for our pipettes. [Link to Pipettes page]

Biosafety Cabinet

Biosafety cabinet [Link to Equipment overviews page]

PCR hood

PCR hood [Link to Equipment overviews page]

Qubit

Qubit [Link to Qubit overviews page]

Post-PCR equipment

[Link to Post-PCR equipment page]

Reagent Storage

Links to different types of reagents we have and storage conditions (frozen, fridge, room temp) [Link to Reagent storage and inventory page]

Waste Disposal

[Link to Waste disposal page]

DNA sampling & extraction protocols

Plant DNA Barcode Reference Library

We are often asked for guidance and assistance building plant DNA reference libraries. The strategies and logistics involved will inevitably differ depending on the goals of a research project, conditions in the field, and access to resources such as existing herbarium collections and experts on the local flora. Here we provide some general suggestions based on experience. Our general Protocol for Collecting Plant Vouchers and DNA Samples is available HERE.

  • The google doc is viewable by anyone with a Brown University email address; contact TRK for access/edit permissions.
  • The doc refers to two metadata sheets. One for is collecting herbarium voucher specimens that can be submitted for mounting, archiving, and digitization at Brown University; this can be requested from the Director of the Brown Herbarium or by emailing TRK. The second is for collecting plant DNA barcode samples that is compatible with the barcode of life data systems, which can be downloaded from boldsystems.org.

Zymo Soil & Fecal Mini Kits

This is the kit we use for most of our fecal DNA research. Please note that the company can change (and has historically refined) the protocol. For this reason, the wiki does not recapitulate information contained within the protocol. You must refer to the protocol from the box you are using to ensure you have the correct version. We simply provide helpful tips for organizing and maintaining the highest standards for your workflow. [Links to Zymo soil fecal kit documents]

MPBIO Plant DNA extraction and barcoding

[Link to protocol from Ian]

PCR protocols

Standard Primer Sets

Below are some useful PCR recipes using reagents that we have (or have had) in the lab for various primer sets and sample types. Please report optimizations and troubleshooting to the lab so that we can make adjustments as needed. These protocols should work, but please do think critically about how they should be adjusted to the needs of your particular project.

Mammal Barcoding

1. Mitochondrial DNA

Plant Barcoding

1. Chloroplast DNA. These protocols mostly use NEB polymerase, simple thermocycling protocols, and sequence well with standard 'pre-mixed' options at GeneWiz. The matK and trnH-psbA protocols could be converted from Phusion for more regular use.

Invertebrate Barcoding

1. Mitochondrial DNA

Illumina Primer Sets

  • Illumina sequencing primers should be engineered with appropriate overhangs for incorporation of sequencing adapters according to URI's standard workflow and the Nextera protocol.
  • Metabarcoding protocols should employ high quality polymerases. This means they should minimally be hot-start polymerases (inactive initially at room temp). We have often used Amplitaq Gold II. Other polymerases that also include proof-reading and high-fidelity features are desirable.

Plants

The trnL-P6 marker is one that we have used a lot for dietary DNA analyses. Most of our projects rely on primers g/h but we dabble in using primers c/h. We have used several polymerases for various projects, but it is critical to always maintain consistency within projects. The recipes below correspond to several current/recent projects. Discuss with TRK before starting any new projects, as we are currently moving to a new approach.
Primers g/h:

  • Amplitaq Gold II polymerase: trnL-P6 g/h protocol: Amplitaq. This protocol has been used for Kenya small mammal diet analyses and most of our earlier large mammal diet analyses. Note that this is a 15 uL recipe, which we used for small mammal diets; our standard for large mammal diets ranged from 12.5 uL to 20 uL (see, e.g., Kartzinel et al. 2015; Kartzinel et al. 2019). Note also that this protocol worked really well with the shorter MID-tags that we used in our published work, but did not readily transfer to the longer overhangs required for a Nextera protocol; we could get strong amplification based on primers engineered to have these long overhangs by boosting the Mg concentration to 3.5 mM, but as of mid-2021 we have not generated sequence data based on amplicons generated using this level of Mg.
  • Platinum Taq polymerase: trnL-P6 g/h protocol: Platinum. This protocol has been used for Yellowstone large mammal diet analyses projects on elephants and giraffes in Kenya. We have found it works well with the Nextera overhangs on these primers.

Primers c/h: [Beth and TRK should update the trnL-P6 section as part of a "module"; note also that we now use Platinum protocols for Kenya g/h]

Invertebrates

  • The IN16STK primers are designed to amplify invertebrate 16S rRNA (DNA sequences) from vertebrate diets. We have both the original IN16STK and the more recent degenerate IN16STK-mod primers available in the lab.
  • [Ian, Beth, and TRK should update based on experience with Caroline and Courtney]

Bacteria

The 16S V4 marker is one that we have used for several microbiome studies. It's critical to always maintain consistency within projects, but we have used several approaches across projects over the years. The recipes below correspond to several current/recent projects. Discuss with TRK before starting any new projects, as we are currently moving to a new approach.
Primers 515f/806r:

  • We have used an Accuprime Mastermix protocol to analyze the gut microbiomes of small mammals in Kenya and large mammals in Yellowstone: 16S-V4 rRNA protocol. These primers are designed to work with the Nextera library prep method.
  • The long Earth Microbiome...

Gel protocols

[Link to Gel Room Protocols page for overview and specifics]

Targeted Sequencing

Targeted sequencing, also called Sanger sequencing, is the protocol we use to sequence single-band/single-product amplicons. We perform sequencing in both directions.

Amplicon clean up

[Link to submitting Sanger sequencing samples]

Other targeted sequencing steps

Pending decisions to standardize and streamline sequencing protocols for the lab; multiple options may ultimately be available, depending on the urgency and scale of a sequencing project...

  • Chain-terminator (BigDye) reactions:
  • Submission for sequencing.
  • Sequence assembly and analysis.
  • Submitting to BOLD
  • Submitting to GenBank

Illumina MiSeq At URI

Illumina sequencing

[Link to Illumina sequencing protocols page for basics on meta barcoding options]