RESTRICTION DIGESTION AND ANALYSIS OF LAMBDA DNA KIT

I. Objectives:
● Understand restriction enzymes, their function, and their utilization as biotechnology tools.
● Learn to separate DNA fragments on an agarose gel using electrophoresis.
● Generate a standard curve from a series of DNA size standards.
● Estimate DNA fragment sizes from agarose gel data.
● Understand how to use a restriction digestion map to identify a sample DNA.

II. Introduction:
DNA splicing, the cutting and linking of DNA molecules, is one of the basic
tools of modern biotechnology. The basic concept behind DNA splicing is to remove a
functional DNA fragment - let’s say a gene - from the chromosome of one organism and to
combine it with the DNA of another organism to study how the gene works. The desired result
of gene splicing is for the recipient organism to carry out the genetic instructions provided by
its newly acquired gene. For example, certain plants can be given genes for resistance to pests
or diseases, and in a few cases to date, functional genes have been given to people with non-
functional genes, such as those who have a genetic disease like cystic fibrosis.
In this laboratory activity, your task will be to cut (or digest) lambda DNA,
the genomic DNA of a bacterial virus, and then determine the size of the DNA pieces using a
procedure known as gel electrophoresis. This involves separating a mixture of the DNA
fragments according to the size of the pieces. Once this is accomplished, you will compare
your pieces of DNA with pieces of DNA whose size is already known.
Of the DNA fragments that are produced, imagine that one piece in particular represents a
specific gene. This gene can code for any number of traits. But before it can be given to a
recipient organism, you must first identify the gene by using gel electrophoresis.
Your tasks:
● Cut lambda DNA into a series of fragments using restriction enzymes.
● To separate and sort a large group of DNA molecules according to their size.
● To determine the size of each molecule separated by gel electrophoresis.
You will be provided with lambda DNA and three different restriction enzymes. The DNA
restriction analysis that you are about to perform is fundamental to a variety of genetic
engineering techniques, including gene splicing, DNA sequencing, gene localization, forensic
DNA matching, or DNA fingerprinting.
Before you begin, it might be helpful to review the structure of DNA and the activity of
restriction enzymes (see FULL MANUAL).
Activity before class
Watch a clip for the introduction of:
● How to make an agarose gel for DNA electrophoresis;
● How to load a sample to wells of a gel;
● How to run a gel in an electrophoresis chamber; and
● How the gel is stained and visualized

III. Materials:
Shared apparatus:
1.0% agarose gel with RedSafe Nucleic Acid
Chemicals and Staining Solution ● Prepared by instructors.
Reagents TAE 1X Buffer ● 4 groups/ 1 gel
DNA Marker (M)
Vortexer 2 groups/1 equipment
Mini centrifuge (spin down) 3 groups/1 equipment
Apparatus and
Heat block 1 equipment for all groups
Equipment
Horizontal electrophoresis system 1 system for all groups
Gel imaging system 1 system for all groups

Each group will be provided with:

Samples and 0.3 µg/µL λ DNA 20 µL
Reagents PstI restriction enzyme 2 µL
EcoRI restriction enzyme 2 µL
HindIII restriction enzyme 2 µL
10X Green Buffer (restriction buffer) 20 µL
Deionized water 200 µL
Micropipette 20 µL 1 piece
Micropipette 10 µL 1 piece
Apparatus and Cooling rack 1 piece
Equipment Microcentrifuge tube rack 1 piece
250 mL beaker 1 piece Waste cup
Marker pen 1 piece
Consumables Micropipette tips 200 µL (yellow tips) 1 box
Micropipette tips 10 µL (white tips) 1 box
1.5 mL microcentrifuge tube 1 pack
IV. Procedures:
The DNA you will be provided with has been extracted from a bacteriophage - a bacterium-
invading virus. The virus is known as lambda and is often written as “λ”. You will be working
with three different restriction enzymes, also called endonucleases. These are referred to as
PstI, EcoRI, and HindIII.

1. Obtain the cooling rack that contains the three enzyme solutions, lambda DNA, and
restriction buffer. Important note: Keep all the reagents in the cooling rack and
minimize the time of handling the reagents at room temperature.
2. Obtain four 1.5 mL microcentrifuge tubes and label them with your group’s number
and the following letter:

o Tube 1: L (lambda DNA)

o Tube 2: P (PstI lambda digest)
o Tube 3: E (EcoRI lambda digest)
o Tube 4: H (HindIII lambda digest)

3. To tube L, add 15 µL of deionized water. To tube P,E,H, add 14µL of deionized water
to each tube
4. To each tube, add 2 µL of 10X Green Buffer
5. To each tube, add 3 µL of λ DNA
6. Add 1µL of PstI to tube P
7. Add 1µL of EcoRI to tube E
8. Add 1µL of HindIII to tube H

Important note: DO NOT add enzyme into the tube labelled “L”. Place the tubes into the
cooling rack immediately after adding enzyme.

After step 8, the volume and content of each tube should be as follows:
Deionized 10X Green λ DNA PstI EcoRI HindIII Total
Tube st nd rd th
water (1 ) Buffer (2 ) (3 ) (4 ) (4th) (4th) volumn
L 15 µL 2 µL 3 µL - - - 20 µL
P 14 µL 2 µL 3 µL 1 µL - - 20 µL
E 14 µL 2 µL 3 µL - 1 µL - 20 µL
H 14 µL 2 µL 3 µL - - 1 µL 20 µL

9. Tightly cap each tube. Gently vortex (50% power) and briefly centrifuge (spin down)
all samples. Put the tubes back on the cooling rack and bring them to the heat block.
10. Place the tubes in the heat block and incubate for 30 minutes at 37°C. Put your cooling
rack in the freezer (-25oC).
 11. Then, heat all samples at 80°C for 5 minutes. Obtain your cooling rack from the freezer
and return the samples to the cooling rack – this will result in better separation of the
DNA bands.
Load 20 µL of each sample into separate wells in the gel chamber in the following order
(from left to right). Use a fresh tip for each sample.

Lane Sample
1 M, DNA marker (pre-loaded into the gel by your instructor)
2 L, uncut lambda DNA
3 P, PstI lambda digest
4 E, EcoRI lambda digest
5 H, HindIII lambda digest

12. Carefully place the lid on the electrophoresis chamber. Connect the electrical leads into
the power supply, red to red and black to black.
13. Turn on the power and run the gel at 110V for 30 minutes.
14. When the electrophoresis run is complete, turn off the power and remove the top of the
chamber. Carefully remove the gel and tray from the gel box. Be careful — the gel
is very slippery.
15. Observe and capture the DNA bands under UV illumination.

V. Lab Report: (short report)

● Short introduction (10 points): follow instructions for the full report but it should be
written not exceeding 1/3–1/2 of a page.
● Material and Procedure (10 points): Some sentences to describe briefly what had
been performed in the experiment which followed the protocols of … (the precise Kit
should be indicated)
● Results and discussion: (70 points) (ALL PAGES REFER TO FULL MANUAL)
o Direct gel examination (instruction in pages 37-39). Fill in the table on page 38
for the final report. (15 points)
o Creating a Standard Curve (instruction in pages 40-43). You need to generate
the curve using Semilog Graph Paper (page 42) and fill in the Table in page 43
for final report. (15 points)
o Answer the 4 questions in page 44 for final report (40 points)
● Writing and layout (10 points).
o File type: You should submit your lab report as a pdf/doc file, all other types are
not allowed.
o References: References should be in Harvard style. Refer to guidance from
library.
o Format: Times New Roman, 12pt, 1.5 spaced, left margin 2.5 cm, top, right and
bottom margin: 2cm.