TITLE: DNA  Quality Control by Agarose Gel Electrophoresis
AUTHORS: Benjamin Schwessinger

[DESCRIPTION]
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:
 
 
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:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846332/
https://en.wikipedia.org/wiki/Agarose_gel_electrophoresis
https://www.youtube.com/watch?v=ZDZUAleWX78
 

[BEFORE_START]
You must study the protocol carefully before you start. If anything is unclear post questions directly here on protocols.io.

[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.

[STEPS]
SECTION: Week 5: DNA agarose gel electrophoresis
1. 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.
SECTION: Week 5: DNA agarose gel electrophoresis
2. You will receive your ITS PCR strip tubes of 8 with PCR reaction from last week 4. 
The strip will contain the 5 reaction plus negative control for the ITS reaction. 

SECTION: Week 5: DNA agarose gel electrophoresis
3. You will receive your diluted DNA stock solutions @ 10ng/ul for TG1 to TG5 from last week. 
SECTION: Week 5: DNA agarose gel electrophoresis
4.1. Label a two new strip tubes of 8 with PCR or DNA and the respective sample names TG1-TG5 + neg or TG1-TG5. 
SECTION: Week 5: DNA agarose gel electrophoresis
4. Now you will prepare all samples to be ready to be loaded onto the agarose gel.
SECTION: Week 5: DNA agarose gel electrophoresis
4.2. Pipette 5 uL of each PCR reaction or DNA stock [10ng/ul] solution into the correctly labelled strip tube of 8.
SECTION: Week 5: DNA agarose gel electrophoresis
4.3. Add 1 uL of gel loading dye to each sample. This will be 11 samples in total. Six PCR samples and five DNA samples.
Close all tubes carefully once you added the loading dye.
SECTION: Week 5: DNA agarose gel electrophoresis
4.4. 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.
SECTION: Week 5: DNA agarose gel electrophoresis
4.5. Spin down the content of the PCR strip tubes in the small benchtop spinners that take PCR strip tubes of 8. Collect all liquids at the bottom of the tube.
SECTION: Week 5: DNA agarose gel electrophoresis
5. Now you can proceed to loading the samples onto the agarose gel
SECTION: Week 5: DNA agarose gel electrophoresis
5.1. Note down the sample order number on how you indent to load the samples on the agarose gel. For example, DNA ladder; ITS TG1 to TG5, negC; DNA TG1 to TG5; DNA ladder. 
SECTION: Week 5: DNA agarose gel electrophoresis
5.2. 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.
SECTION: Week 5: DNA agarose gel electrophoresis
5.3. Load 5 ul the DNA ladder and your samples as noted down. 
SECTION: Week 5: DNA agarose gel electrophoresis
6. Once all samples are loaded from both research groups, add the lid, connect the power block and set for 1hr @ 100 V.
SECTION: Week 5: DNA agarose gel electrophoresis
7. After 1hr, open the lid and take out the gel.
SECTION: Week 5: DNA agarose gel electrophoresis
8. Image the gel as instructed by the demonstrator.
SECTION: Week 5: DNA agarose gel electrophoresis
9. 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:

20230206_BSPS_PCRGel_2.jpg

Pictures that do not follow this naming convention will not be shared with the class.


SECTION: Week 5: DNA agarose gel electrophoresis
10. 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.