A collection of blog posts connected to my teaching on biomedical sciences and biochemistry degrees. All views and opinions expressed are my own, and not connected to my past, present or future employers.
In this video, I look at Nucleic Acid Hybridisation and how it is the underlying principle for several lab techniques, such as PCR (Polymerase Chain Reaction), dot blots, colony blot hybridisation, chromosome in situ hybridisation (FISH), microarrays, Southern and Northern blotting, and CRISPR/Cas9 gene editing.
How do scientists determine the size of DNA bands on an agarose gel? In this guide, I will walk you through the step-by-step process of calculating the size in base pairs of a DNA band on an agarose gel.
Blog Bonus: Free information sheet summarising the video and the steps - download.
Introduction
When working in a lab and running an agarose gel, you may need to determine the size of the DNA fragment, and this information may be crucial for various biological research applications.
This approach is also described in the following video:
Setting Up the Experiment
Imagine you have loaded a DNA ladder with known sizes in one lane and your DNA sample with an unknown size in another lane of the gel and you get a result that looks like this when the gel has been run.
Before you can calculate the size of your DNA band, you must first label the gel and collect data to create a calibration curve.
Data Collection and Analysis
By measuring the distances the DNA bands in the ladder (see below) have moved and plotting the log values of their sizes against the distances travelled in millimetres (or you can do it in pixels), you can create a calibration curve. This curve will help you accurately determine the size of the DNA band in your unknown sample.
The image below shows the gel and the data table for the plot.
From the table, you plot the calibration curve.
Calculating the Size of the DNA Band
After plotting the calibration curve (above) and identifying the distance your unknown band has travelled, you can use the curve to determine the size of the DNA band in base pairs. By following a simple formula involving logarithms, you can convert the log value to the actual size in base pairs.
Conclusion
Calculating the size of a DNA band on an agarose gel requires careful data collection, analysis, and interpretation. By following the steps outlined in this guide, you can confidently determine the size of DNA fragments in your samples.
In this video, I examine the step-by-step process of cloning DNA into plasmids and address common challenges faced in the lab. I start by preparing the DNA and then move on to using restriction enzymes like EcoRI and HindIII. I explain the importance of choosing the correct enzyme pairs to prevent self-ligation and ensure the correct orientation of the insert. I also cover the blue-white selection method to verify successful cloning and discuss using different vectors for larger DNA segments.
In this video, I give a brief introduction to the subject of DNA cloning.
DNA cloning is an important lab skill that all life and biomedical science students should possess. In the video, I provide a summary of in vivo and in vitro cloning, along with the key steps, tools and methods you would use.
I have just posted a video that looks at the complex processes of gene regulation and protein production within cells. In the video, I explore six key stages where cells regulate protein synthesis, ensuring precise protein production according to cellular needs. These stages include:
Transcriptional Control: The initiation of RNA production and the involvement of transcription factors.
RNA Processing: The conversion of pre-messenger RNA to mature mRNA, including splicing and editing.
Transport and Localisation: The transportation of mRNA to the correct cellular locations for protein synthesis.
Translational Control: Regulation of mRNA translation within the cellular environment.
RNA Degradation: Mechanisms that determine the lifespan of mRNA molecules.
Protein Regulation: Various methods cells use to control protein activity, such as chemical modifications and localisation.
This is the second of two videos on how cells make proteins using DNA. In the first video, I looked at the first step, which is making the messenger RNA (mRNA) a process called transcription - Transcription - making proteins from DNA - the mRNA.
In this video, I will guide you through the process of producing the protein from the mRNA, also known as translation. I will look at the coding problem (how many mRNA bases do you need to code from an amino acid), the number of reading frames in a DNA molecule, and how the cell produces protein from the mRNA.
In this video, I will guide you through the first steps in the process of producing RNA from DNA, also known as transcription. In the next video, we will take the next step and examine how we produce the protein from mRNA.
The video looks at the five steps of mRNA production:
initiation — activators bind upstream, often thousands of bases upstream, of the gene. The activators assemble the required proteins (the mediator, chromatin remodelling complex, the RNA polymerase and transcription factors) at the TATA box, which is a DNA sequence close to the gene
production — DNA is transcribed into the pre-messenger RNA
five prime capping — the five prime end of the pre-messenger RNA is capped with some modified nucleotides
splicing — introns are spliced out of the pre-messenger RNA to leave just the exons (exons provide the sequence for the protein)
three prime polyadenylation — addition of a polyadenylated tail to the three prime end of the pre-messenger RNA to give the final mature messenger RNA molecule
The video not only looks at mRNA production but also introduces the idea of non-coding RNA (ncRNA), which are RNA molecules that do not encode proteins. ncRNAs are essential in regulating gene expression and various cellular processes. For example:
transfer RNA (tRNA) — involved in protein synthesis
ribosomal RNA (rRNA) — involved in protein synthesis
microRNA (miRNA) — control gene expression
small interfering RNA (siRNA) — control gene expression
long non-coding RNA (lncRNA) — diverse functions
circular RNA (circRNA) — gene regulation
Piwi-interacting RNA (piRNA) — genome protection
enhancer RNAs (eRNAs) — modulate gene activity
Finally, I cover how to write out DNA sequences, the means of the terms sense and antisense strands, and what we mean by upstream and downstream when talking about DNA and RNA molecules.
