Jan 21, 2026
Protocol: Routine Authentication of Drosophila using RFLP Markers
- Lauren McCann1,
- Natalia Rivera Rincón1,
- Emma Saurette1,
- Melika Ghasemi Shiran1,
- Laurie Stevison1
- 1Auburn University
- Drosophila Stock Validation
Protocol Citation: Lauren McCann, Natalia Rivera Rincón, Emma Saurette, Melika Ghasemi Shiran, Laurie Stevison 2026. Protocol: Routine Authentication of Drosophila using RFLP Markers. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7n3mklwz/v1
Manuscript citation:
Shiran MG, Bailey NP, McCann L, Rivera-Rincón N, Saurette E, Stevison LS. 2026. Efficient RFLP-based Protocol for
Routine Authentication of Drosophila. microPublication Biology. 10.17912/micropub.biology.001949.
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: September 20, 2025
Last Modified: January 21, 2026
Protocol Integer ID: 227731
Keywords: Restriction fragment length polymorphism, routine authentication of drosophila, rflp markers stock validation, using rflp markers stock validation, drosophila, dna extraction, gel electrophoresi, ecor1 enzyme, dmel-57
Funders Acknowledgements:
NIH
Grant ID: R35GM147501
NIH
Grant ID: P40OD018537
Abstract
Stock validation is an important factor in scientific research; by authenticating the resources used there is an increase of the quality of research to be performed. This protocol was designed to validate ten stocks of D. melanogaster and four stocks of D. pseudoobscura in the DGRP. Designed for DMEL_42, DMEL_57, DMEL_217, DMEL_357, DMEL_391, DMEL_399, DMEL_437, DMEL_491, DMEL_508, and DMEL_810_ of D. melanogaster; in addition to MV2-25, TreeLine, Flagstaff14, and Flagstaff16 of D. pseudoobscura. Following sample collection, DNA Extraction, PCR Amplification, EcoR1 Enzyme, and Gel Electrophoresis you will be able to confirm the identities of your samples in comparison to the expected results.
Materials
Step 1: Sample Collection
- Fly stocks of interest
- CO₂ to anesthetize flies
- Standard Dissecting Microscope
- -20°C freezer
- 96-well plate with sealer or PCR strip tubes with caps
- Labels
Step 2: DNA Extraction (Gloor and Engels 1992)
- Squish Buffer (SB)
- 100 µL 1M Tris-HCl pH 8.2
- 20 µL 0.5M EDTA
- 50 µL 5M NaCl
- 9.9 mL dH₂O
- Stored at -20°C
- 20 mg/mL proteinase-K solution
- Thermocycler
Step 3: PCR Amplification
- DNA template to be added to master mix
- Master mix
- Promega GoTaq‱ G2 Green Master Mix (Promega M7822)
- Upstream primer (10µM)
- Downstream primer (10µM)
- Nuclease-free water
- New 96-well plate with new sealer and labels
- Thermocycler
Step 4: ECOR1 Enzyme
- Nuclease-free water
- 10X NEBuffer EcoR1/SspI
- DNA sample from the previous step
- EcoR1 enzyme
- Purple loading dye
- New 96-well plate with new sealer and labels
- Thermocycler
Step 5: Gel Electrophoresis
- Agarose gel
- 1x TAE Buffer
- EtBr (Ethidium Bromide)
- Microwave
- Erlenmeyer flask
- Gel Electrophoresis system
- Well combs
- DNA Ladder
Step 6: Azure Pictures
- Azure Imaging Systems (AZI200-01 - AZI600-01)
Troubleshooting
SAMPLE COLLECTION
Prepare and label your tubes for stocks of interest, collecting 2 females of each stock.
KEY
DMEL_42 (42), DMEL_57 (57), DMEL_217 (217), DMEL_357 (357), DMEL_391 (391), DMEL_399 (399), DMEL_437 (437), DMEL_491 (491), DMEL_508 (508), DMEL_810 (810), MV2-25 (MV), TreeLine (TL), Flagstaff14 (FS14), Flagstaff16 (FS16)
Anesthetise flies with CO₂, once all flies are anesthetised, continue to Step 3.
Pour flies onto CO₂ table of your microscope, then sort out 2 females and place into pre-labeled tubes (see step 1 chart). Then move onto next variety, making sure to not mix up any stocks.
- 2 female samples of each stock variant
- 1 fly per tube
Place flies in -20°C freezer one day prior to following DNA extraction step
DNA EXTRACTION
Create a master mix by combining Squishing Buffer with Proteinase-K Solution for the following 50µL reaction, amplifying by 1 plus the number of samples
- 49.5 µL Squishing Buffer
- 0.5 µL Proteinase-K Solution
- This solution must be mixed fresh
Briefly vortex your master mix until homogeneous
Draw up 50 µL master mix solution and begin to stab one fly repeatedly with the tip of the pipette, slowly release the master mix solution to moisten the fly so it will stick to the tube.
After the fly is mashed (~20 seconds and the liquid has turned red) eject any remaining master mix liquid into the tube over the carcass. Replace pipette tip and repeat for the rest of the samples.
*Keep on ice until all preps are done
When finished with all samples, run the following thermocycler protocol as follows:
- 37°C for 30 minutes
- 95°C for 2 minutes
- Volume 50 µL
When thermocycler is finished, samples can be placed in -20°C freezer or continue to next stage.
- Preps are often good for a year (or longer) if amplifying small DNA fragments, though repeated freeze/ thaw will shorten its lifespan.
PCR AMPLIFICATION
Select Primers
DPSE → 2 markers
DMEL → 4 markers
2 Samples Per Stock
1 DPSE sample -> will be allocated into 2 seperate wells with appropriate primers
1 DMEL sample -> will be allocated into 4 seperate wells with appropriate primers
1- 96 well plate for samples. Colors represent the different primers assigned to each sample
Prepare master mixes for each marker according to the following quantities for a single 12.75 µL reaction based on 1 plus the number of samples to provide a negative control.
6.5 µL Promega GoTaqGreen
0.5 µL Upstream primer (10µM)
0.5 µL Downstream primer (10µM)
5.25 µL Nuclease-free water
Pipette 11.75 µL of the master mix into wells as assigned (see Diagram in Step 12)
Add 1 µL of DNA sample into appropriate wells, again following the diagram in Step 12.
- A multi-pipette can be used to transfer DNA samples
Seal well plate and run the thermocycler protocol for touchdown PCR as follows:
- 2 min. 95 °C
- 10 Cycles of:
- 30 sec. 95 °C
- 30 sec. 65 °C (decreasing by 1°C each cycle)
- 30 sec. 72 °C
- 20 Cycles of:
- 30 sec 95°C
- 30 sec 55°C
- 30 sec 72°C
- 2 min. 72°C
- Hold 4 °C
After the program is done, the samples can be stored in -20°C freezer, or continue to next stage.
ECOR1 ENZYME PROCEDURE
In a new 96 well plate, add the following into each well IN ORDER AS LISTED ENSURING TO ADD THE ENZYME LAST
- 3.4µL Nuclease-free water
- 1µL 10X NEBuffer EcoR1/SspI
- 5µL DNA sample from the previous step
- 0.5µL EcoR1 enzyme
Seal well plate and run thermocycler program for ECOR1 as follows:
- 60 min 37°C
- Volume 10 µL
After the program is done, add 2µL of purple loading dye into each well to stop the reaction.
Once dye is added, the samples can be stored in the -20°C freezer, or continue to next stage.
GEL ELECTROPHORESIS
Making the gel
- Measure 2g of agarose and add to an Erlenmeyer flask
- Add 100mL of 1x TAE buffer into the flask and swirl
- Microwave this solution in 30 second increments and swirl until color is clear and all powder is dissolved
- Let your gel mixture cool before adding Etidium Bromide, you should be able to touch the flask and feel warmth without burning
- Add 5µL of EtBr to flask and swirl
- Ready to pour into gel tray
Pour prepared gel into the gel tray, with the well comb in place, and let gel solidify
Once gel is solidified: Remove the tray and leave the gel in the gel box (electrophoresis unit), with the wells on the black side
- DNA will run to the red side
Add 1x TAE buffer to cover the gel in the gel box
- Buffer must correspond to the buffer in the gel or it will not work
Add 5µL of the ladder into the first well and Pipette 5µL of each DNA sample into corresponding wells
- When dispensing, make sure the pipette tip does not stab the gel and that all the sample goes into the well. Keep track of the order in which the samples are placed so they can be identified once the gel has run.
Place cover on and attach power source
- DNA sample will run toward the positively charged elecctrode (red) since DNA is negatively charged
Set voltage and time accordingly (100v for 30 min)
Ensure everything is running smoothly, small bubbles will appear
Once gel is run, take photos as needed and analyze results
AZURE PICTURES
Turn on the machine, place the gel into the tray and close the door.
Select Nucleic Acid dye: Ethidium bromide
Take the picture, edit as needed to view the bands
Discard gel and clean tray
ANALYSIS
Compare your gel results to the guide below, ensuring your stamples performed as anticipated.
Protocol references
Gloor GB, Engels WR (1992) Single-fly DNA preps for PCR. Drosoph. Inf. Serv. 71:148-149
GoTaq‱ Green Master Mix (M712) Protocol
R.H. Don, P. T. Cox, B.J. Wainwright, K. Baker, J. S. Mattick, ‘Touchdown’ PCR to circumvent spurious priming during gene amplification, Nucleic Acids Research, Volume 19, Issue 14, 25 July 1991, Page 4008, https://doi.org/10.1093/nar/19.14.4008