Nov 13, 2024
7500 Sex marker screening protocol EFGL
- 1Eagle Fish Genetics Lab
Protocol Citation: EagleFish GeneticsLab 2024. 7500 Sex marker screening protocol EFGL. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn98zml5d/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: November 08, 2024
Last Modified: November 13, 2024
Protocol Integer ID: 111827
Abstract
This is a generalized protocol that Eagle Fish Genetics Lab (EFGL) uses to prepare samples for a genetic sex screening on the ABI 7500. It provides steps on normalizing samples, prepping different master mix recipes, and using the 7500 software v2.3 to complete a 7500 run.
Materials
2X TaqMan Universal PCR Master Mix Applied Biosystems (ThermoFisher Scientific)Catalog #4318157
MicroAmp™ Optical 96-Well Reaction PlateThermo FisherCatalog #4316813
MicroAmp™ Optical Adhesive FilmThermo FisherCatalog #4311971
Ultrapure Distilled, Nuclease Free WaterContributed by users
Equipment
Applied Biosystems 7500 and 7500 Fast Real-Time PCR Systems
NAME
Applied Biosystems
BRAND
4351107
SKU
Some guidelines before starting:
Some guidelines before starting:
This is a generalized protocol for working sex assays in EFGL.
- If you’re working on a newly designed assay, there could be changes to setup, reagent concentration, etc. Ultimately, it shouldn’t stray too far from this.
EFGL normalizes all sample DNA to 10ng/µl before performing any 7500 runs.
For an optimal representation of results, each plate setup should ideally have a 50/50 split, or as close as possible of known males and females.
- If all samples are of unknown sex, run known sex samples as “positive controls” for comparison. Consult biologist on which samples are ideal as controls.
- Include at least 2 NTCs per assay per plate
EFGL's default 7500 experiment type is “Genotyping”.
Setting up project and samples
Setting up project and samples
Obtain sample list and assay(s) needed from lead biologist/data coordinator. Samples may have been
pre-extracted (stored in -80 °C ), or extract them if necessary before proceeding.
Use the 7500 Quantification and Input File v1.1 spreadsheet in your project folder, to generate quantification and input files of your tray(s).
The spreadsheet template is setup so that it'll match the plate layout as you do each 7500 runs.
Enter in the project name, and tray information to keep track of samples for that tray.
Enter sample names in “Sample Information” tab starting from cell C7.
- Enter known phenotypic sex for the samples in column D. OK to leave blank if unknown.
- NTC wells have been set to wells G12-H12.
Save new file for each tray you’ll run.
Repeat steps 3 to 3.3 until all samples have been mapped out.
To have a visual layout, head to the “7500 plate map” within spreadsheet
You can also start copying and pasting each tray map to the 7500 PCR labsheets v1.2 spreadsheet to obtain master mix recipe. OK to do this after normalization as well.
Depending on sample size, determine which quantification protocol (HTX plate reader or Qubit HS) would best fit.
Note
Qubit is recommended/easier if:
- Total sample size is less than 80, or
- There’s a lot of cherry picking from several trays for each plate to run
Recommended to quantify and normalize all samples prior to starting the 7500 runs. PLAN accordingly as this section is the most time consuming
Qubit HSFrom 25 to 41 steps
Prep Qubit master mix (199 µL of buffer to 1 µL of dye per sample) for 4 or 5 samples first.
In a Qubit assay tube, add 198 µL of Qubit master mix and 2 µL of DNA per sample. Gentle vortex, quick spin and incubate for 00:03:00 at Room temperature prior to read
If Qubit machine says, “too high”, dilute the remaining samples to 1:10 dilution. If not, proceed as normal for the remaining samples.
(Skip if no dilution is required) In a new unskirted PCR plate, add 1 µL of DNA to 9 µL of water. Heat seal, vortex, quick spin and use this diluted DNA for Qubit quantification.
Using the same 7500 quantification and input spreadsheet, enter in the quantified DNA concentration in "Qubit + Norm" tab. It has been formulated to normalize all samples to 10 µg/µL at 15 µL final volume.
If using diluted DNA, change the dilution factor number in cell D2 to obtain the actual DNA quantification.
In column D (DNA tray well), enter in the original DNA well position. Print out this sheet to begin normalization.
In a new unskirted PCR plate, add the calculated water + DNA sample to each well. Heat seal, vortex and quick spin .
Expected result
Your samples are now all normalized to 10 µg/µL at 15 µL
Proceed with the remaining tray(s), and save all normalized trays till end of project.
Once all DNA samples have been normalized, in the same spreadsheet, head to the "7500 plate map" tab for each tray.
Copy each tray map and paste it to the respective tray in the 7500 PCR labsheets v1.2 spreadsheet. Print individual sheets if necessary.
Going back to the 7500 Quantification and input spreadsheet. Click on the "SNP Sample Sheet" tab and save it as .txt (Tab delimited) file on a flash drive.
Bring the flash drive with you to the lab, to import samples to the 7500 computer for running the experiment later.
EFGL uses 2 types of assays. Taqman assay, and IDT assays.
Taqman assays come ready to go with all primers and probes pre-optimized by Thermo Fisher. While IDT assays are ordered in separate components, 2 primers (forward and reverse), and single or double probes.
Note
Master mix recipes listed are based on default concentration/volume EFGL uses for genotyping experiments along with the default cycling conditions.
Taqman assays27 steps
Thaw and gently vortex Taqman assay. As the tube itself doesn’t fit in our vial centrifuge, gently tap tube on benchtop to bring assay down to the bottom of tube.
In a 1.5mL vial, prep the master mix. To account for pipetting error, add 2 additional samples.
Gently vortex normalized DNA tray, quick spin down .
Use a 0.1 mL combitip to dispense 9 µL of master mix into the ABI plate wells.
Using a p10 multi-channel pipette, add 1 µL of normalized DNA to the respective wells
IMPORTANT! Add DNA to the side of the well to avoid creating bubbles – 7500 runs are sensitive to bubbles.
Seal the ABI plate with a MicroAmp film by using a Kimwipe to press down on seal to avoid smudges/scratches on film.
DO NOT VORTEX. Place a kimwipe at the bottom of the centrifuge plate holder in the centrifuge to avoid any black rubber flecks sticking to the ABI plate and do a quick spin down for about 10-15 seconds.
Load the ABI plate onto the 7500 machine. Orient your plate such that well A1 is at the top left corner of the loading tray, if not, your results will be flipped.
Set up 7500 Run
Set up 7500 Run
Turn on both 7500 machine and the computer. Plug in flash drive into computer after startup.
Launch 7500 software v2.3, and log into a profile.
(Skip to step 15, if assay isn't new)
Add new SNP assay to library before proceeding with 7500 runs.
Go to Tools, and click on "SNP Assay Library..."
Select "New...", and enter the SNP assay name and details. Here's an example:
Click "OK" to save assay. Now this assay is available for future runs.
Click on Advance Setup in the Set Up column of the home page.
On the Experiment properties page, make the following edits:
- Edit experiment name - following project name would be ideal
- Select "Genotyping" for the type of experiment. Click "OK" to the pop-up. Leave everything as default. (Not shown in picture, at the very bottom of page, leave "Pre-PCR Read", "Amplification" and "Post-PCR Read" boxes checked)
- Click on "Plate Setup" to import samples.
On the Plate Setup page, click on "File" on the top left corner, and select "Import.." from dropdown.
Import the saved .txt file from your flash drive. Your samples should auto-populate in the tray map on screen.
If there's an error on import, likely the .txt file was not saved correctly. Save/overwrite .txt file and try again.
In the "Assign SNP Assay(s) to the Selected Wells." box, select the assay that you'll be running with.
- Click on the SNP assay name "template", it'll turn purple/blue upon clicking (EFGL's input file template has defaulted the SNP assay as "template", thus it needs to be switched to the correct assay)
- Click on "Edit ", select "Delete SNP Assay"
3. Once deleted, click on “Add Saved SNP Assay” and select the appropriate SNP assay(s) in pop up window. Then, click “Add selected SNP assay(s)”
Once you have the right SNP assay(s) show, select all samples to run on that assay.
- To select all samples, click on the small white square on top left corner of tray map, or select the samples individually for separate assays.
- Check the "Assign" box to assign that assay to the selected samples
If you run a partial plate, clear all empty wells.
- Select all empty wells, right click on mouse, and select “Clear”.
- This prevents flags/errors on empty wells after the run has been completed.
Select both NTC wells from plate layout, and click on dropdown box under “Task”
- Select "Negative Control"
Next, on the Experiment Menu. Click on "Run Method".
Edit reaction volume from 50 µL to 10 µL
Make any changes to the cycle conditions, if necessary. EFGL have used the default cycling conditions on most of our assays.
Once all changes are made, click "Save".
Click on the green "START RUN" button at the top right corner to begin run. (there can be a bit of a lag, so don't click it more than once)
Default run time is approximately 1.5-2 hours.
Collect results
Collect results
Once the run has completed, the screen will be on the Allelic Discrimination plot page.
A successful run would likely show distinct and tight clusters. In some instances, the plot will require manual calling of each sample to reflect the right calls.
Take a screenshot of plot, and export all data to flash drive. Upload plot image and raw data files to your project folder.