Lab Report: DNA Extraction and Electrophoresis

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1. Introduction

Deoxyribonucleic acid (DNA) extraction is a crucial process for isolating DNA from cell nuclei. This process involves various steps, including alkaline lysis and DNA purification. Alkaline lysis is commonly used to purify plasmid DNA. Purification is typically carried out using the phenol-chloroform method due to its safety and efficiency. Polymerase Chain Reaction (PCR) is a technique that requires a nuclease-free environment and precise protocol execution, involving denaturation, annealing, and extension steps. Agarose gel electrophoresis is employed to separate DNA molecules based on size, with factors such as agarose concentration, running buffer, voltage, and staining method affecting the migration velocity of DNA fragments (Elkins, 2012; Turner, 2005; Linacre & Tobe, 2013; Maddocks & Jenkins, 2016; Kieleczawa, 2005; Pelt-verkuil, Belkum & Hays, 2008; Walker & Rapley, 2009; Lottspeich & Engels, 2018).

2. Materials and Methods

2.1 Plasmid DNA Extraction: Alkaline Lysis Minipreps

Plasmid DNA extraction was performed as follows:

1 ml of E.

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coli culture was transferred to a microcentrifuge tube and centrifuged at maximum speed for 60 seconds.
The culture was removed with a micropipette while retaining the pellet at the bottom of the tube.

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This step was repeated with an additional 1 ml of the culture into the same tube.
The tube was vortexed for 2 minutes and placed on ice for 5 minutes.
200 μl of denaturing solution was added to the tube, mixed by inversion for 5 seconds, and put on ice for 5 minutes.
150 μl of cold renaturation solution was added to the tube and mixed by inversion for 20 seconds. The tube was placed on ice for 5 minutes.
The tube was then centrifuged at maximum speed for 5 minutes, and the supernatant was transferred into a new tube.
2x volume of cold 96% ethanol was added to the tube, mixed by vortexing for 2 minutes, and placed on ice for 2 minutes.
The nucleic acid mixture was centrifuged at maximum speed for 5 minutes at room temperature, and the supernatant was removed.
The pellet was washed by adding 1 ml of 70% ethanol, spun for 1 minute, and air-dried for 30 minutes.
The pellet was resuspended in 50 μl of TE buffer.
The DNA concentration was measured using a nanodrop spectrometer in DNA mode.

2.2 Polymerase Chain Reaction (PCR)

The PCR reaction mixture was prepared by calculating the necessary dilutions of DNA and the amount of components 1, 2, 3, and 5. The reaction mixture was then prepared, and 20 μl of the mixture and the diluted DNA were added to a PCR tube.

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The tube was labeled, spun down, and stored in the fridge for later analysis.

2.3 Nucleic Acid Electrophoresis (Agarose Gel Electrophoresis)

The following steps were followed for nucleic acid electrophoresis:

1% agarose gel was prepared by mixing agarose in 1X TAE buffer inside a flask. The flask was covered with plastic wrap and heated in a microwave for 2-3 minutes and then cooled for 5 minutes.
The gel casting tray was prepared by sealing the end of the tray with tape and inserting the well-forming combs.
Nucleic acid dyes were added to the cooled agarose solution, and the flask was mixed by swirling.
The agarose solution was poured into the casting tray, allowed to solidify, and then placed into the gel tank with the comb located near the negative electrode.
The DNA samples and ladder were reconstituted with 6X loading buffer.
6 μl of the DNA ladder was loaded in the leftmost lane of each row, and 10 μl of each PCR tube was added into separate wells.
The electrophoresis tank's lid was closed, and the power supply was turned on to 10V/cm. After running for 4-6 minutes, the power supply was turned off, and the gel was photographed.

3. Results

NanoDrop spectrophotometry was used to determine the DNA concentration and purity of samples 1 and 2. The DNA concentration of sample 1 and 2 were 994.0 ng/µl and 904.5 ng/µl, respectively. To achieve the recommended concentration, sample 1 needed a 20x dilution, while sample 2 required an 18x dilution. The A260/A280 ratio for sample 1 was 1.79, and for sample 2, it was 2.04 (Table 1).

Sample	Concentration (ng/µl)	Extracted DNA Yield - A260 (10mm)	Purity (A260/A280)
Sample 1	994.0 ng/µl		1.79
Sample 2	904.5 ng/µl		2.04

The gel electrophoresis results showed that the DNA ladder contained 14 bands with DNA fragment sizes ranging from 10,000 to 250 base pairs. The log of the ladder was calculated, and a linear equation was derived.

3.1 DNA Ladder

Bands	Migration Ladder Bands (cm)	DNA Size (bp)	Log Ladder
1	5.8	10,000	4
2	6.1	8,000	3.903
3	6.4	6,000	3.778
4	6.8	5,000	3.699
5	7.2	4,000	3.602
6	7.4	3,500	3.544
7	7.9	3,000	3.477
8	8.2	2,500	3.398
9	8.8	2,000	3.301
10	9.2	1,500	3.176
11	10.2	1,000	3
12	10.4	750	2.875
13	11.2	500	2.699
14	11.9	250	2.398

3.2 Log Base Pairs vs. Migration Distance

The linear equation obtained from the log of base pairs vs. migration ladder bands measured in cm was: y = -0.2394x + 5.3553.

3.3 Actual Size of The Base Pairs in The Sample

The migration distance for PCR products A (number 14) and B (number 15) were 10.6 cm and 10.4 cm, respectively. Using the linear equation, the actual size of the base pairs was calculated (Table 3).

Experimental Sample	Migration PCR Production (cm)	Calculated Y	Antilog Y
14	10.6	2.818	657.14
15	10.4	2.866	733.74

4. Discussion

Samples 1 and 2 exhibited relatively high purity with A260/A280 ratios of 1.79 and 2.04, respectively, where a ratio of 1.80 indicates good purity. Both samples had DNA concentrations exceeding 50 ng/µl, necessitating dilution with nuclease-free water. Sample 1 required a 20x dilution, while sample 2 needed an 18x dilution. Gel electrophoresis demonstrated a migration pattern, with smaller DNA fragments traveling farther and larger fragments remaining closer to the top of the gel.

The DNA ladder, containing known and standardized base pair sizes, was used as a reference to verify the actual size of DNA fragments. The linear relationship between the log of base pairs and migration distance was established through the log ladder. The linear equation y = -0.2394x + 5.3553 allowed for the calculation of the actual size of base pairs for PCR products A and B.

Possible sources of error in gel electrophoresis include inaccurate measurements using a ruler, variations in print quality affecting the visibility of DNA ladder bands, pipetting errors, and sample contamination during preparation.

5. Conclusion

The experiment successfully determined the actual size of base pairs in the samples based on DNA ladder migration patterns. Possible errors were identified, including measurement inaccuracies, print quality, pipetting mistakes, and sample contamination.

6. References

Elkins, K. (2012). Forensic DNA biology. Oxford: Elsevier.
Turner, P. (2005). Instant notes molecular biology. New York, NY: Taylor & Francis.
Linacre, A., & Tobe, S. (2013). Wildlife DNA analysis. West Sussex, U.K.: John Wiley & Sons.
Maddocks, S., & Jenkins, R. (2016). Understanding PCR. [S.l.]: Academic Press.
Kieleczawa, J. (2005). DNA sequencing. Sudbury, Mass: Jones and Bartlett.
Pelt-verkuil, E., Belkum, A., & Hays, J. (2008). Technical Aspects and Principles of PCR Amplification. Dordrecht: Springer.
Walker, J., & Rapley, R. (2009). Molecular biology and biotechnology. Cambridge: Royal Society of Chemistry.
Lottspeich, F., & Engels, J. (2018). Bioanalytics: Analytical methods and concepts in biochemistry and molecular biology. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co.

Lab Report: DNA Extraction and Electrophoresis. (2024, Jan 02). Retrieved from https://studymoose.com/document/lab-report-dna-extraction-and-electrophoresis