Download Week 2. DNA isolation and PCR

Supplemental File S6: DNA isolation and PCR analysis of
as1 and as2 mutants
INSTRUCTOR PRE-LABORATORY PREPARATION
The instructor will need tissue from wild type, as1 and as2 mutant Arabidopsis plants for the DNA
isolation. If the plants were saved from the week 1 activities, the best material to use is 6-8 flower buds. If
the instructor chooses to use leaf tissue from the saved plants, 2-3 young leaves that have not fully
expanded are the best source of DNA. If the instructor prepared seedlings for this week’s activities, 2-3
seedlings should be used for each DNA isolation. If the instructor froze young leaf tissue from the week 1
activities, the tissue should be removed from the freezer only when extraction buffer and grinding of the
tissue can immediately occur. The DNA isolation protocol is a modification of Dellaporta et al. 1983 (1)
but any simple plant DNA isolation protocol will work. The PCR primers (see supporting file S1 or S13
for primer sequences) will need to be ordered and prepared at a concentration of 5 M in advance of the
laboratory. During the laboratory session, students will need tips, tubes, Pellet PestlesTM, microcentrifuge,
thermocycler, scalpels and forceps. Each pair of students should get either a wild type, as1 or as2 mutant
plant. Each pair of students is also given either AS1 or AS2 primers. A sample guide for distributing the
plants and primers to twelve pairs of students is shown in table 1.
Group
Plant genotype
1 and 2
Wild type
3 and 4
Wild type
5 and 6
as1 mutant
7 and 8
as1 mutant
9 and 10
as2 mutant
11 and 12
as2 mutant
Table 1-Suggested setup for distribution of plants and PCR primers.
PCR primers
AS1
AS2
AS1
AS2
AS1
AS2
The activities are designed to use a 2X PCR master mix (Fisher Scientific FERK0171) but could be
modified to use other PCR reagents. As written, the protocol asks the students to perform the calculations
to determine how much of each reagent goes into the PCR. This step could be removed if desired. The
amounts of each reagent is shown in the table 2.
Component
amount in
L
12.5
1
1
1
2X PCR mix
5 M primer 1
5 M primer 2
genomic
template DNA
water
9.5
(total volume)
25
Table 2-Amounts of reagents required for each PCR.
The students are asked to make predictions about the sizes of the PCR products they expect from these
experiments. Prior to the laboratory, the instructor will need to give the students the AS1 and AS2 primer
sequences and the wild type genomic sequences of the AS1 and AS2 genes (see supplemental file 13).
One goal of the in-laboratory discussion is for the students to understand how to predict the size of the
wild type AS1 and AS2 PCR products. During the laboratory, the students will be asked to fill in table 3.
The students will only be able to accurately predict the size of the mutant PCR products if they were
given the background information on AS1 and AS2 (Supplemental file S3) and understand the mutations
in as1 and as2. At this point in the activity, I focus on the “DNA sequence expected to be wild type or
mutant” column. It is not unusual for students to state that a PCR product is expected to have a mutant
sequence but then give the size of the wild type PCR as the expected length. This is an indication that the
students have quite grasped what a mutation is. At this point in the activity I merely note which groups
have the PCR sizes incorrect. I will ask these groups later in the series of activities to revisit their
calculations.
DNA isolated from:
PCR primers used
wild type
AS1
wild type
AS2
as1 mutant
AS1
as1 mutant
AS2
as2 mutant
AS1
as2 mutant
AS2
Table 3-Size of the expected PCR products.
DNA sequence
expected to be wild
type or mutant?
Wild type
Wild type
Mutant
Wild type
Wild type
Mutant
How long (in base
pairs) will the PCR
product be?
840
615
839
615
840
602
STUDENT PRE-LAB PREPARATION
Instructions to be given to students: The goal of this week’s laboratory is to isolate DNA from as1, as2
and wild type Arabidopsis plants and use this DNA to PCR amplify the AS1 and AS2 genes. PCR is a
laboratory modification of natural cellular DNA replication. To help you understand how PCR works,
please watch the following video prior to attending the laboratory
(https://www.youtube.com/watch?v=2KoLnIwoZKU). You also need to watch a second video which
demonstrates wild type cellular DNA replication (https://www.youtube.com/watch?v=I9ArIJWYZHI).
Once you have watched the videos, write a few paragraphs that clearly compare and contrast the process
of PCR with cellular DNA replication.
Analyzing the sequence of genes you want to PCR amplify is required in order to determine if the PCR
worked. The instructor will give you the AS1 and AS2 primer sequences and the genomic sequences of the
AS1 and AS2 genes. Find the AS1 primer sequences in the AS1 gene and calculate the expected PCR
product size. Repeat this process with the AS2 primers and gene sequence.
Finally, as part of the laboratory, you will need to write a paragraph, appropriate for the materials and
methods section of a paper, explaining the PCR process. To help you understand how materials and
methods sections are written, prior to the laboratory, you need to find a primary literature article that used
PCR to collect data and contains a description of the PCR protocol in the Materials and Methods sections
of the paper. Bring this paper to the laboratory.
INSTRUCTOR NOTES FOR IN-LABORATORY DISCUSSION
I begin the in-laboratory discussion by asking the students to share their compare and contrast paragraphs
of PCR and DNA replication with a student from another group. Then, I lead a class discussion that
begins with having the students volunteer similarities and differences between PCR and DNA replication.
I like to show the PCR video (https://www.youtube.com/watch?v=2KoLnIwoZKU) in the laboratory and
provide my own commentary to ensure that students understand the PCR process.
As a class, I then ask the students to volunteer the expected sizes of the AS1 and AS2 PCR products they
calculated in their pre-laboratory assignment. Some students will not have found the reverse PCR primer
because they do not understand that the reverse primer sequence needs to be reversed and complemented
before they can find and exact sequence match of the primer in the gene sequence. I often have to remind
students that DNA consists of two complementary strands and have to explain that it is common to
present only one strand of DNA sequence when a gene sequence is given. The other common error that
students make is forgetting to include the PCR primers in the length of the PCR product. To correct this
misconception I often replay the PCR video and specifically point out that the primer is part of the PCR
product. This discussion will help the students complete the table in task 3 of the laboratory handout, but
some students will still have difficulties understanding that in a mutant plant only the mutant gene has a
sequence that is different from wild type; all other genes have a wild type sequence. For example, in as2
mutants the AS2 gene is mutated and produces a PCR product 13 bp smaller than the wild type AS2 gene
but the AS1 gene produces a wild type sized PCR product.
Finally, I have the students volunteer how the authors of primary literature papers present a summary of
the PCR in Materials and Methods section of the paper. As the students volunteer information about the
primary literature article they found for the pre-laboratory assignment, I make sure that students
understand that the primer sequences and the PCR conditions must be presented. After this initial
discussion, I give the students 5 minutes to look at protocol in their student laboratory handout and ask
them to compare and contrast the summary of PCR from the materials and methods section of a primary
literature article and their protocol. As a group we have a short discussion where I make sure that students
understand that when they are writing the materials and methods section of a paper they cannot simply
copy and paste the protocol from the student handout.
SAMPLE STUDENT LABORATORY HANDOUT
Introduction: To analyze the DNA sequence of the AS1 and AS2 genes, you will isolate genomic DNA
from wild type Arabidopsis plants and the as1 and as2 mutants. The genomic DNA will serve as the
template in PCR. The primers used in the PCR will specifically amplify the AS1 and AS2 genes.
Ultimately the 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.
Task 1: 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 2: 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 5M 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
2X PCR mix
5 M primer 1
5 M primer 2
genomic
template DNA
water
(total volume)
amount in
L
1
25
Step 2. 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 3: 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
Fill in the table:
DNA isolated from:
PCR primers used
wild type
wild type
AS1
AS2
DNA sequence
expected to be wild
type or mutant?
How long (in base
pairs) will the PCR
product be?
as1 mutant
as1 mutant
as2 mutant
as2 mutant
AS1
AS2
AS1
AS2
Task 4: Write a brief summary of how the PCR was setup as it would appear in the Materials and
Methods section of a primary literature article.
References
1.
Dellaporta SL, Wood J, Hicks JB. 1983. A plant DNA minipreparation: Version II. Plant Mol
Biol Report 1:19–21.