Background

We typically order in whole plates (96 samples) for each individual loci (ITS1F and ITS4). The advantage here is two-fold. First, for every two plates that we order, we only need to make a single sample plate. Our sequencing provider is able to take an ITS1F sample and and ITS4 sample from the single 96-well plate you send in. Secondarily, primer plates will not have to be created to go along with each shipment. 1.5 mL microcentrifuge tubes of primer will suffice. In sum, for every shipment, we will be making a single 96-well sample plate and sending in one 1.5 mL tube (1000 ul) of 3 uM solution of each primer (Our sequencing provider has a stock in-house of primers for our project).

Plate Preparation

Supplies Needed:

1. Empty 96-well plate.
2. Aliquot of molecular water. 
3. Multichannel pipette/tips for 30 uL transfers. 
4. Pipette/tips for 10 uL transfers.
5. Pipette/tips for 20 uL transfers.
6. Caps for 96-well plate.
7. PCR Product
8. Plate Preparation Template

Protocol:

1. Print off and fill out the Plate Preparation Template. It is best to fill this out beforehand with the samples you would like to utilize for the plate being prepared.

2. Put 30 uL of molecular water into each cell in the 96 well plate. A multichannel pipette is usually the best for this. 

3. For each sample that had a strong band on the gel, include 10 uL of sample into the cell.  For each sample that had a weak band on the gel, include 16 uL of sample into the cell. 

4. Cap off the plate. We typically cap off the plates with strip caps.

5. Label your plate with the appropriate plate number. Lab Name - Date - Internal Code. Ex - UWL-NAMP014

Primer Preparation

For each sample plate that is produced, one 1.0 mL of each primer in a 3 uM concentration will need to be provided. Our sequencing provider has a stock of these already prepared for our project.

Protocol:

1. Pull stock primers from -80, thaw, gently spin.
2. Label eppi tube with primer name and "3uM."
3. WITH FILTER TIP add 970 uL of molecular grade water.
4. WITH FILTER TIP add 30 uL of primer. 
5. Invert gently to mix.

Prepare Shipment

Typical packaging is the sample plate and two primer tubes in a ziplock bag. Label the exterior of the bag with your plate numbers.


Plates prepared and ready for shipment.

**Ship your sample plates FedEX 2-Day Express or overnight utilizing our account number.**

Ship your samples Monday - Wednesday. We do not ship on Thursday or Friday to reduce the chance our sample plates may arrive, but sit in the box over the weekend.

Prepare the Plate Spreadsheet

Below you will find a downloadable template for the Plate Submission Spreadsheet. This is the form that is electronically submitted to our sequencing provider that outlines and describes the order being sent to them. Fill out this spreadsheet and email it to info@mycoflora.org. We will upload it to the online sample submission platform.

About North American Mycoflora Project

NAMP is a 501(c)(3) non-profit organization. We are working towards a single goal - the development of the first comprehensive mycoflora of North America. This project is a consortium of citizen scientists and professional mycologists performing a biological survey of all the macrofungi that occur in North America.

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