Feb 09, 2025
DNA Quality Control of PCR products and native DNA by Agarose Gel Electrophoresis
- 1Australian National University
- BIOL3106/6106
Protocol Citation: Benjamin Schwessinger 2025. DNA Quality Control of PCR products and native DNA by Agarose Gel Electrophoresis. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9r58zv3e/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: February 09, 2025
Last Modified: February 09, 2025
Protocol Integer ID: 119846
Abstract
This protocols is part of the ANU Biosecurity mini-research project #1 "Plant Pathogen Diagnostics: Visuals, subcultures, and genomics".
You will be provided four pots of 3-4 week old wheat plants that have been infected with different wheat pathogens. Each pot has been infected with one major pathogen. You will not know which pot has been infected with which pathogen. However, you will be provided a compendium of 10-15 wheat pathogens that will guide you to identify the infective agent for each treatment group. The fifth treatment group will be uninfected wheat plants which will be clearly identified. You can use treatment group #5 as negative control for your experiments.
In total, each group will obtain five pots each:
| A | B | |
| Treatment group 1 | Unknown infective agent | |
| Treatment group 2 | Unknown infective agent | |
| Treatment group 3 | Unknown infective agent | |
| Treatment group 4 | Unknown infective agent | |
| Treatment group 5 | Uninfected control |
This specific protocol is a step by step guide to assess DNA quality via agarose gel electrophoresis. Agarose gel electrophoresis lets you assess the size of DNA molecules. In addition, you can estimate DNA concentrations and impurities of RNA.
During agarose gel electrophoresis DNA gets linearised. Larger molecules take longer to migrate through the gel when migration is driven by a electric potential at the specific pH of the buffer. When running a standard DNA ladder with bands of known molecular size, one can compare the DNA size of ones DNA or PCR sample with the known standards.
The final goal is to achieve the following:
- To assess DNA length of molecules in total DNA stock samples and for all PCR reactions.
- To approximately estimate relative DNA concentrations.
- To test if negative PCR controls did not amplify anything as appropriate.
- To test if whole DNA extracts contain RNA in addition to DNA.
- Select samples from three research groups to be sequenced in the practical of week 6.
This protocol is applicable for week 5.
Protocols progress overview:
- Week 5 Run a 1% Agarose Gel electrophoresis for all samples of each research group.
Some useful explainers and resources:
Image Attribution
The icon was created with BioRender.com.
Guidelines
You must have read, understood, and follow the health and safety instructions provided in the "Overview Mini-Research Project #1 BIOL3106/6106" provided on Wattle (ANU learning portal).
You must have signed and returned one copy of the "Student Safety Declaration Form For Practical Class Work" before starting any laboratory work.
You must have read and understood the Hazard Sheets (Risk assessment) of all chemicals listed bellow in the "Safety Warnings" section. These Hazard Sheets are provided on Wattle as part of the "Overview Mini-Research Project #1 BIOL3106/6106" document.
Materials
As always you need to bring a lab notebook, a printed version of this protocol, and a pen to record your adventures in the lab.
Consumables and culture material needed:
- Pre-cast 1% Agarose Gel in TBE
- PCR strip tubes
- Gel Loading Dye, Purple (6X); 20 uL total. https://www.nebiolabs.com.au/products/b7024-gel-loading-dye-purple-6x#Product%20Information
Equipment needed:
- Mini benchtop spinner for PCR strip tubes of 8.
- Voltage block.
- DNA gel electrophoresis chamber
Safety warnings
This protocol does not require any hazardous substances.
You need to wear safety equipment at all times including lab coats, gloves, and safety goggles when handling chemicals and biological agents. While the major biological agents used for the wheat infection are pathogens commonly found in Australia, you must treat them as they were infective agents of general concern. Treat them with care. Do not remove them from the laboratory. Do not spread them via clothing. Use a dedicated notebook and pen to make notes during the mini-research project. Do not put anything into your mouth while in the laboratory. Wash your hands each time you leave the laboratory.
Before start
You must study the protocol carefully before you start. If anything is unclear post questions directly here on protocols.io.
Week 5: DNA agarose gel electrophoresis
Week 5: DNA agarose gel electrophoresis
1h 48m
1h 48m
You will load all DNA and PCR samples into one lane of a 1% agarose gel each. You will share your gel with another research group as each gel has two rows.
You will receive your ITS and 16 PCR strip tubes of 2x8 with PCR reaction from last week 4.
Each strip will contain the 5 reaction plus negative control for the ITS or 16S reaction, respectively.
5m
You will receive your diluted DNA stock solutions @ 10ng/ul for TG1 to TG5 from last week 4.
2m
Now you will prepare all samples to be ready to be loaded onto the agarose gel. The PCR reactions are ready to load as they already contain loading dye (redish), while we still need to prepare the native DNA stock solutions.
Label a new strip tubes of 8 with DNA and the respective sample names TG1-TG5.
5m
Pipette 10 uL of each DNA stock [10ng/ul] solution into the correctly labelled strip tube of 8.
5m
Add 2 uL of gel loading dye to each sample. This will be 5 samples in total.
Close all tubes carefully once you added the loading dye.
5m
Now mix the samples with the gel loading dye by flicking them briefly with your finger. Make sure all tubes are closed properly before flicking.
2m
Spin down the content of the strip tubes in the small benchtop spinners that take strip tubes of 8. Collect all liquids at the bottom of the tube.
2m
Also spin down the content of your PCR strip tubes (16S and ITS) before proceeding to loading the agarose gel.
Now you can proceed to loading the samples onto the agarose gel.
Note down the sample order number on how you indent to load the samples on the agarose gel. For example,
For the 17 samples plus two DNA ladder/markers.
DNA ladder; ITS TG1 to TG5, negC; 16S TG1 to TG5, negC; DNA TG1 to TG5; DNA ladder.
5m
You will share one gel with another group. Each group will load their samples either in the top or the bottom row. Note down which row you are using and co-ordinate with your paired research group.
Load 5 ul the DNA ladder and 10 ul of your samples as noted down in step 5.1.
10m
Once all samples are loaded from both research groups, add the lid, connect the power block and set for 1hr @ 100 V.
1h
After 1hr, open the lid and take out the gel.
Image the gel as instructed by the demonstrator.
5m
Make sure to label your pictures as follows so we can share them online in class and you can clearly identify your pictures.
YYYYMMDD_ResearchGroupName_PCRGel_PictureNumber
For example:
20250206_BSPS_PCRGel_2.jpg
Pictures that do not follow this naming convention will not be shared with the class.
2m
Now you have captured the DNA quality and size distribution. We will discuss results in class and select the samples of three research groups for metabarcode sequencing and total DNA sequencing in week 6.