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Lab 2 Lab Manual

This document provides instructions for a laboratory exercise on isolating DNA from a Drosophila larva using a spin column method and analyzing the extracted DNA through gel electrophoresis. Students will extract DNA from a fruit fly larva, purify it using a commercial kit, and observe the results on a gel to learn about DNA isolation techniques and principles.

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makabigail7
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43 views

Lab 2 Lab Manual

This document provides instructions for a laboratory exercise on isolating DNA from a Drosophila larva using a spin column method and analyzing the extracted DNA through gel electrophoresis. Students will extract DNA from a fruit fly larva, purify it using a commercial kit, and observe the results on a gel to learn about DNA isolation techniques and principles.

Uploaded by

makabigail7
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
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BIOL2313 Genetics Laboratory 2023-2024

Exercise 2
DNA isolation from a Drosophila larva

DNA isolation is an extraction method that aims to purify DNA from a tissue/organism, in
which DNA is separated from cell membranes, proteins, and other cellular components by
using physical and/or chemical means. In 1869, Friedrich Miescher achieved DNA isolation
for the first time. Afterwards, many different protocols have been developed by different
scientists to isolate DNA for different purposes. One of the methods named “phenol-chloroform
method” was developed and is one of the most traditional but widely compatible methods used
in different research nowadays. Meanwhile, another method named “spin column-based
method” was widely adopted today because it does not include the usage of any hazardous
chemicals and is an effective, reliable, and consistent method. This method is a solid phase
extraction method that purifies nucleic acids in a short period of time. It relies on the nature of
nucleic acid that it binds to the solid phase of silica under certain conditions.

Regardless of which isolation methods, the principal behind DNA isolation is more or less
similar and consist of the following three major parts: (1) disruption of cellular and nuclear
membranes; (2) separation and isolation of DNA from other cellular components including
lipids, proteins, and other nucleic acids; and (3) purification and concentration of DNA.

In this exercise, you are going to extract DNA from a Drosophila larva using the spin column-
based nucleic acid purification and analyze the results by gel electrophoresis.

Learning outcomes:

After this lab session, you should be able to:


1. Understand the rationale and principle of DNA isolation
2. Extract DNA from Drosophila larvae
3. Perform DNA isolation with commercial kits
4. Read laboratory protocols
5. Perform gel electrophoresis
6. Be familiar with the do’s and don’ts when performing DNA experiments.
7. Understand the safety precautions in gel electrophoresis

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BIOL2313 Genetics Laboratory 2023-2024

Procedures:
DNA isolation
1. Aliquot 180 ul of digestion buffer (DB) in a microcentrifuge tube.
2. Label the tube with your group number and your name initials.
3. Clean the forceps, spatula and homogenizer with 75% ethanol.
4. Take out a scoop of Drosophila medium with a spatula and spread it in double distilled
water on a petri dish.
5. Isolate one larva and rinse it in double distilled water to remove other debris.
6. Use a clean forceps to put the larva into the digestion buffer.
7. Homogenize the larva with a plastic homogenizer thoroughly. (Do not dispose the
homogenizer)
8. Add 20 ul of proteinase K (PK) to the tube.
9. Add 20 ul of RNase A (RA) to the tube.
10. Incubate the tube at 55°C for 30 min.
11. Add 200 ul Lysis/binding buffer (LB) and 250 ul of 100% ethanol (ETOH) to the tube.
12. Vortex the tube for 5 sec.
13. Label a spin column with your group number and your name initials.
14. Transfer the solution to a spin column with a collection tube.
15. Spin the tube for 10,000 rpm for 1 min.
16. Remove the collection tube* and replace it with a new one.
*Contained the flow-through that does not have DNA as DNA is already bound to the silica membrane
17. Add 500 ul Wash Buffer 1 (WB1) to the spin column.
18. Spin the tube for 10,000 rpm for 1 min.
19. Discard the collection tube and replace it with a new one.
20. Add 500 ul Wash Buffer 2 (WB2) to the spin column.
21. Spin the tube for 10,000 rpm for 1 min.
22. Discard the collection tube and replace it with a new one.
23. Spin the tube for 10,000 rpm for 30 sec.
24. Discard the collection tube and replace it with a new MICROCENTRIFUGE TUBE
(Label the microcentrifuge tube first with your group number and your name initials).
25. Add 20 ul of elution buffer (EB) to the center of the silica membrane of the spin column.
26. Incubate for 2 min.
27. Spin the tube for 10,000 rpm for 1 min.

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BIOL2313 Genetics Laboratory 2023-2024

28. Proceed the sample to gel electrophoresis.

Gel electrophoresis
29. Prepare a 1% agarose gel for electrophoresis (Performed by demonstrator).
30. Take 15 µl of the isolated DNA and mix it with 3 µl of 6x sample loading buffer in a PCR
tube.
31. Load the samples onto a 1% agarose gel (together with the 1kb DNA marker, negative
control and positive control) and run at 120 V for 30 minutes.
32. Take a photo of the gel under UV illuminator.
33. Observe the size of the DNA band.

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BIOL2313 Genetics Laboratory 2023-2024

Reference figures:

Figure 1. Life cycle of Drosophila melanogaster (A) Life cycle of Drosophila melanogaster
modified from (Weigmann et al., 2003) at 25°C. (B) Big and small vial normally used for
breeding and keeping of Drosophila flies and larvae. (C) Arrangement of adult Drosophila fly
(female), pupa and late 3rd instar larva. Genotype w1118 . Scale bar = 1 mm.

Figure 2. Isolated DNA band observed from a gel electrophoresis, with the marker (ladder)
indicating the band size (basepair).

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BIOL2313 Genetics Laboratory 2023-2024

Reference information:

PureLink™ Genomic DNA Mini Kit Protocol:

https://www.thermofisher.com/document-connect/document-
connect.html?url=https://assets.thermofisher.com/TFS-
Assets%2FLSG%2Fmanuals%2Fpurelink_genomic_mini_man.pdf

Lab 4 activities:

DNA isolation – 120 min

Gel electrophoresis – 30 min

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