HOW MUTATIONS IN THE AS1 AND AS2 GENES CAUSE

MUTATIONS IN PLANTS
At least one member of your group will need a laptop in order to complete this lab.
There will be a learning catalytics quiz at the beginning of lab. The quiz will
potentially cover the basics of the lab described below as well as basic laboratory
solution/dilution calculations.
Introduction: During the next four weeks, the class will explore the AS1 and AS2
genes of Arabidopsis. The overall goal of these experiments is to understand how
changes at the DNA level can result in a mutant phenotype. The goals of the week 1
activities are to analyze the phenotype of as1 and as2 Arabidopsis mutants, isolate
DNA from wild-type, as2 and/or as2 mutants, and PCR amplify the AS1 and AS2 genes.
Ultimately the AS1 and AS2 PCR products will be sequenced and you will analyze the results to
determine how the DNA sequence of the AS1 and AS2 gene varies in wild type, as1, and as2 plants.
During the PCR setup, record what plant your group is isolating DNA from (wild type, as1 or as2) and
what PCR primers (AS1 or AS2) your group is adding to the reaction.

Before you begin this laboratory, download and save this student handout to a computer. Complete the
laboratory by following the instructions provided. When you are finished with the laboratory, upload your
completed handout to the course management system. Make sure you also email the completed lab to all
of your lab partners. Everyone will need this data to write a lab report.

Task 1: Use your cell phone to take pictures that clearly show the leaf phenotypes
of the wild type and mutant Arabidopsis plants. You will use these images to create
a publication quality figure. Make sure that each figure has a ruler in the picture.
The ruler should be placed in a region where it can be cropped out of the image.
This will allow you to make a scale bar in your figure.
Task 2: Measure the petioles of the two most mature leaves from the wild type and
mutant plants. Starting with the wild type Arabidopsis plant, carefully remove two of
the most mature leaves from the plant by holding the leaf blade with forceps and
using a razor blade to cut the petiole as close to the stem of the plant as possible.
Measure the petiole from where it joins the base of the leaf to where it was cut from

the stem. Record this measurement in the table below. Repeat this process for the
as1 and as2 mutants. Once you have completed your groups table below, add your
data the class spreadsheet.
Genotype/leaf #

petiol
e
lengt
h
(mm)

wild type/1

.8 cm

wild type/2

.8 cm

as1/1

.2cm

as1/2

.3cm

as2/1

.9cm

as2/2

.9cm

Task 3: Isolate DNA from Arabidopsis plants. The following protocol is based on the method by
Dellaporta et al. 1983.
Step 1. Collect a small amount of plant tissue. The instructor will clarify what type of tissue you should
use and how much tissue you will need. Place the tissue in a 1.5 mL microcentrifuge tube.
Step 2. Add 400 L of extraction buffer (200 mM Tris-Cl, pH 7.5; 250 mM NaCl; 25 mM EDTA; 0.5%
SDS).
Step 3. Use a blue Pellet PestlesTM to mash the tissue in the buffer. The plant tissue should mostly
disintegrate. BE CAREFUL NOT TO SPLASH OUT ANY SOLUTION WHEN YOU ARE GRINDING.
Step 4. Centrifuge the tube at maximum speed for 10 minutes. MAKE SURE THE CENTRIFUGE IS
BALANCED!
Step 5. Place the supernatant in a new 1.5 mL tube and add 300 L of isopropanol; mix by inversion.
Step 6. Centrifuge at maximum speed for 10 minutes.
Step 7. You should see a small pellet (this is your DNA). Carefully discard the supernatant by carefully
pouring or pipetting the liquid and keep the DNA pellet.
Step 8. Add 200 L of 70% ethanol to the pellet.
Step 9. Centrifuge the tube at maximum speed for 5 minutes.

Step 10. Carefully pour off the 70% ethanol; again you can pour but do not lose the pellet!
Step 11. Leave the tube open and allow the pellet to air dry for 5-10 minutes.
Step 12. Add 100 L of TE (10 mM Tris-Cl, pH 8.0; 1 mM EDTA) to the pellet.
Step 13. Carefully pipette up and down until the pellet is dissolved.
Step 14. Spin tube for 1 minute to pellet the cell debris.
Step 15. Keep the tube on ice; this is your template DNA for PCR.
Task 4: Amplify the AS1 and AS2 genes by PCR
Step 1: Calculate the reaction
a) Your reaction will need two PCR primers (the instructor will tell you what primers to use). Each
primer will be given to you as a 5M stock solution. Your final PCR reaction must contain 0.2
M of each primer. The final total volume for the PCR must be 25 L. How much of each primer
will you need to add?
b) You need to add 2X PCR mix. This mix contains the Taq DNA polymerase, Mg++, buffer and dNTPs
required for the reaction. The PCR mix is supplied at a 2X stock concentration. Your final reaction must
contain 1X PCR mix. How much of the 2X PCR mix will you need to add?
c) We will use 1 L of the genomic DNA isolated from Arabidopsis as our template for PCR.
Fill in the table. Have the instructor check your numbers before you set up your reaction.
Component

amount in

2X PCR mix
5 M primer 1
5 M primer 2
genomic

L
12.5
1
1
1

template DNA
water
(total volume)

9.5
25

Step 2. Wait until the instructor tells you to do this step. While you are waiting, you can work on Task 5.
Set up your reaction. Pipette in this order: 1) water, 2) PCR mix, 3) primer 1, 4) primer 2, 5) DNA.

2. Briefly spin the tube.

3. Ask the instructor how to label the reaction and where to place it when you have completed the PCR
setup.
Task 5: Predict the PCR results.
Despite the fact that your group setup only one PCR, each group will analyze the data collected by the
entire class. Some groups isolated DNA from wild type plants, others isolated DNA from as1 or as2
mutants. In addition, the PCR primers that the groups used were different; some groups used primers that
amplify the AS1 gene while other groups amplified the AS2 gene. Overall, the class will PCR amplify two
genes from three different Arabidopsis genotypes resulting in six different reactions (see the table below).
These PCR products will be sequenced and analyzed. Before the PCR products can be analyzed, you need
to predict the size of each PCR product and whether the PCR product will have a wild type or mutant
DNA sequence by filling in the table below. Please see the AS1 and AS2 sequence file for instructions on
how to complete this step.
Fill in the table:

DNA isolated from:

wild type
wild type
as1 mutant
as1 mutant
as2 mutant
as2 mutant

PCR primers used

AS1
AS2
AS1
AS2
AS1
AS2

DNA sequence

How long (in base

expected to be wild

pairs) will the PCR

type or mutant?
mutant
wild
mutant
wild
mutant
wild

product be?
40
39
40

Task 6: Insert your plant images into this Word document (insert the below). Use
Insert Shapes to create a scale bar for each picture. As necessary, use the Picture
Format tools to change brightness/contrast, create borders, wrap text, rotate, crop
and insert a figure legend. Use the help feature in Word and ask the instructor if you
need assistance.
Insert images here:
FIGURE 1
FIGURE 3

FIGURE 2

FIGURE 1. Wild type Arabidopsis having long petioles and broad flat leaves.(yellow
bar indicates 1 cm)
FIGURE 2. AS1 Arabidopsis having long petioles and small wrinkled leaves.(yellow
bar indicates 1 cm)
FIGURE 3. AS2 mutant Arabidopsis having small petioles and small flat leaves.
(yellow bar indicates 1 cm)

Task 7: Write a brief summary of the DNA isolation and PCR as it would appear in the Materials and
Methods section of a primary literature article.
DNA isolation is the process of taking an organism, in this case the leaf of the Arabidopsis plant and
its mutants, add extraction buffer and isopropanol to obtain the DNA pellet, then add to the
appropriate amounts of primers and PCR mix.

MATERIALS AND METHODS

Plant materials: The plant material used for this lab was the Aridopsis AS1
mutant. One leaf was cut from the plant and ground up into 400 microliters
of 200 mM Tris-Cl, pH 7.5 and 250 mM EDTA 0.5% SDS.
Solutions:

Extraction Buffer: 200 mM Tris-HCl (pH 7.5), 250 mM EDTA 0.5% SDS
Ethanol 70%.
AS1 Primer left

H20
AS1 Primer right
Isopropanol.

Extraction procedure: Grind one leaf in 400 microliters of the extraction

buffer until it is mostly dissolved in a 1.5 mL tube. Spin in centrifuge on high
for ten minutes. Add supernatant to a new 1.5 mL tube and add 300
microliters of isopropanol and mix by inversion before centrifuging again for
ten minutes. After draining the supernatant, add 200 microliters of 70%
ethanol and centrifuge for five minutes. Afte draining the supernatant and
allowing the pellet to dry the PCR solution and buffers were added then put
on ice.
PCR amplification: For the isolated DNA sample a PCR was done using a
total solution of 25 microliters consisting of 12.5 microliters of 2X PCR Mix, 1
microliter of 5 microMolar primer 1, 1 microliter of 5 microMolar primer 2, 1
microliter of genomic template DNA, and 9.5 microliters of water.

References
1.

Dellaporta SL, Wood J, Hicks JB. 1983. A plant DNA minipreparation: Version II. Plant Mol
Biol Report 1:1921.