DNA Extraction: A Detailed Process Analysis

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Introduction

‘DNA is an acid in the chromosomes’, found inside of the nucleus and houses the genetic information for making proteins. ‘DNA stands for Deoxyribose Nucleic Acid’ (Collinsdictionary.com, 2019). DNA is a polymer made up of four repeated monomers (A, C, T, G) called nucleotides, each nucleotide has a ribose (pentose) sugar, a nitrogenous base and a phosphate group. ‘The four nucleotides have a different nitrogenous base stuck to their deoxyribose sugar; A contains adenine, T contains thymine, G contains guanine and C contains cytosine’.

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The monomers bind together via the nitrogenous bases. ‘Adenine will only bind to Thymine and Cytosine will only bind to Guanine via hydrogen bond formation’. This forms a ladder like structure which will twist to form a double helix (Khan Academy, 2019).

Materials

E. coli cells

Lysis buffer

Ice bath

Water bath at 60oC

Centrifuge

2ml microcentrifuge tubes

Isopropanol

50% ethanol

Water

UV spectrophotometer

Quartz cuvette microcuvette

Methods

A 0.5 ml culture of E. coli K12 cells were provided.

The E. coli K12 cells were added to a microcentrifuge tube along with 1 ml of lysis buffer, and then mixed well by shaking the tube several times. (The lysis solution contained sodium lauryl sulphate, sodium chloride, sodium citrate, and ethylenediamine tetra acetic acid (EDTA)).

The mixture was incubated for 10 minutes at 60°C. The tube was then cooled in an ice bath until it felt cold to touch. This was left for approximately 7 minutes.

The sample was centrifuged at 13000 g for 1 minute to pellet the cell debris.

1 ml of the liquid was carefully pipetted into a clean microcentrifuge tube.

An equal volume of isopropanol was carefully added - pipetting slowly down the sides of the tube which formed a layer on top of the cell extract. A white, stringy mass appeared above the interface between the cell extract and the isopropanol. This mass was the bacterial cell DNA. If the DNA did not precipitate immediately, the tube was gently swirled whilst avoiding mixing the two layers completely.

The DNA then precipitated for 5 minutes.

The sample was centrifuged again at 13000 g for 5 minutes to pellet the DNA, the liquid was then poured off the liquid into a waste pot. This then left a small white pellet.

The DNA was rinsed off by adding 1 ml 50% ethanol to remove the isopropanol. The DNA became transparent at this point.

This was centrifuged at 13000 g for 1 minute then the leftover liquid was poured into a waste pot.

The DNA was left to air dry for 10 minutes.

The DNA was dissolved by adding 100l of water and incubated at 60oC for 10 minutes.

The DNA was diluted by adding 900l of water and then its absorbance was read at 260nm using the UV spectrophotometer

The concentration of the DNA was then calculated.

Results

The DNA concentration was determined by measuring the absorbance at 260nm, yielding a result of 0.092 abs. Using the equation for real DNA concentration C=A×50, where A is the absorbance and C is the concentration in µg/L, the DNA concentration was found to be 4.6 µg/L.

Calculation

First, we collect the absorbance result which was shown on the UV spectrophotometer at 260nm, which was 0.092 abs. Once we had this number, we then use the extinction coefficient - (A DNA solution of 50 µg/ml has an absorbance at 260nm (A260) of 1.0.) - and multiply 0.092 by 50 in order to get the real DNA concentration as 4.6 µg/L

Discussion

A limitation of the experiment carried out could be that with the UV spectrophotometer, the absorbance is set at 260nm. However, this is particularly close to other setting of 230nm and also 280nm. These absorbencies both measure different types, such as, 230nm measures carbohydrates and 280nm measures proteins. Both proteins and carbohydrates are able to be found throughout the experiment and can still be found in the DNA that has gone through the extraction process. This could mean the readings we got for the DNA sample were inflated due to other substances such as the carbs and proteins, still being left in and falsely scanned by the UV spectrophotometer.

In order to get the absolute correct reading for just the DNA, we would have to calculate the 260/230nm ratio as well as the 260/280nm ratio. This calculation will help us to check the purity of our DNA results. The most common way of finding the purity is by recording the absorbance at 260nm and then dividing that by the reading at 280nm. The standard ratio for the purest DNA is between 1.7 and 2.0. The lower the ratio the more contaminants such as proteins are present (Promega.co.uk, 2019). The 260/230nm ratio is the secondary measure of the purity of the DNA. These values are often higher than those collected from the 260/280 ratio. The calculation is the same, dividing the 260nm result by the 230nm result collected. The common ratio is between 2.0 and 2.2 (Nhm.ac.uk, 2019). If the number calculated is below this, this can mean more carbohydrates are present in the DNA.

References

Nhm.ac.uk. (2019). [online] Available at: https://www.nhm.ac.uk/content/dam/nhmwww/our-science/dpts-facilities-staff/Coreresearchlabs/nanodrop.pdf [Accessed 14 Dec. 2019].

Collinsdictionary.com. (2019). DNA definition and meaning | Collins English Dictionary. [online] Available at: https://www.collinsdictionary.com/dictionary/english/dna [Accessed 14 Dec. 2019].

Khan Academy. (2019). DNA structure and function. [online] Available at: https://www.khanacademy.org/test-prep/mcat/biomolecules/dna/a/dna-structure-and-function [Accessed 14 Dec. 2019].

Promega.co.uk. (2019). How do I determine the concentration, yield and purity of a DNA sample?. [online] Available at: https://www.promega.co.uk/resources/pubhub/enotes/how-do-i-determine-the-concentration-yield-and-purity-of-a-dna-sample/ [Accessed 14 Dec. 2019].