Download Week 3. Gel electrophoresis and Bioinformatics

S7-Gel electrophoresis and Bioinformatics
INSTRUCTOR PRE-LABORATORY PREPARATION
The students will need a 1.5% gel and DNA loading buffer. The protocol is written for twelve groups
of students to load a single gel. I recommend that the instructor prepares at least one extra gel that
can be used to demonstrate gel loading and give the students the opportunity to practice loading a
gel before loading their samples. For demonstration and practice purposes, simply load 6X loading
buffer (30% v/v glycerol, 0.25% w/v bromophenol blue, 0.25% w/v xylene cyanol FF) into the
wells. It is a good idea to have a third gel that can be used as a backup if a student accidentally
damages the class gel while loading their samples.
I typically have students load the AS1 PCR products amplified from wild type, as1, and as2 plants
first, and then have the students load the AS2 PCR products amplified from wild type, as1 and as2
plants. The resulting gels will allow the students to readily compare the size difference between the
AS1 (840bp in wild type plant) and AS2 (615 bp in wild type plants) PCR products. The students can
also see that the AS1 PCR product size difference (1 bp) between the wild type and mutant plants
cannot be resolved under these electrophoresis conditions. The same is usually true of the AS2 PCR
product (13 bp difference between wild type and mutant) although the 13 bp size difference is
sometimes visible.
Part of this activity requires the use of web-based bioinformatics and 3-D protein modeling
resources. I use TAIR (The Arabidopsis Information Resource; www.arabidopsis.org) to gather
amino acid and mRNA sequences and I-TASSER (http://zhanglab.ccmb.med.umich.edu/I-TASSER/;
(1)) to make three-dimensional (3-D) models of the AS1 and AS2 proteins. There are other online
resources available that could be used to retrieve the AS1 and AS2 sequences and to 3-D model the
proteins. The instructor could modify the instructions to use the resources of their choice
Writing instructions for classroom activities that require the use of online resources is challenging
because these resources frequently modify their user interfaces and links. In addition, even from a
single online resource, there are typically multiple ways to obtain the same information. Prior to
using this activity, the instructor should run through the instructions for the online resources and
modify them as needed or desired. The instructions provided here were written in June 2015. If an
instructor prefers to provide the students the sequence data rather than requiring them to retrieve
it, the AS1 and AS2 cDNA, coding-sequences (cds), and protein sequences are provided in
Supporting File S13.
The protocol is written to require the use of I-TASSER to make 3-D models of the AS1 and AS2
proteins. I-TASSER is a free online resource for making 3-D models of proteins. To prevent
overloading this resource, please do not have the student groups individually submit the AS1 and
AS2 amino acid sequence to I-TASSER. Instead, submit the AS1 and AS2 sequences only once, as a
class, to I-TASSER. The submission simply requires setting up an account, copying and pasting the
amino acid sequence into the submission box, providing an email, and clicking the run button.
Currently, each I-TASSER account can submit only one job at a time.
Finally, the answers to the bioinformatics laboratory portion of the activity are as follows.
Answers to Task 3 from student handout:
How many exons are in the AS1 gene? Two
How many introns? One
Is the protein coding sequence interrupted by introns? No
Click on the link to the cDNA sequence of AS1 and copy and paste that DNA sequence here: The cDNA
sequence is provided in Supplemental File S13.
Go back and click on the link to the AS1 CDS sequence (the coding region of the gene from the start to
stop codon). Copy and paste that DNA sequence here: The CDS sequence is provided in Supplemental
File S13.
Go back and click on the link to the protein sequence of AS1 and copy and paste that protein sequence
here: The protein sequence is provided in Supplemental File S13.
Repeat the process to find the AS2 sequences. Note that, because the AS2 gene is alternatively spliced,
there are multiple possible splice variants, i.e. mRNA products (AT1G65620.1, AT1G65620.2,
AT1G65620.3, and AT1G65620.4). For each of the four splice variants, how many exons are present?
How many introns? Is the protein coding sequence interrupted by introns?
AT1G65620.1: Three exons, two introns, no interruption.
AT1G65620.2: Three exons, two introns, no interruption.
AT1G65620.3: Three exons, two introns, no interruption.
AT1G65620.4: Two exons, one introns, no interruption.
Click on the link to the cDNA sequence of AS2 and copy and paste that DNA sequence here: The cDNA
sequence is provided in Supplemental File S13.
Go back and click on the link to the CDS sequence (the coding region of the gene from start to stop
codon) of AS2 and copy and paste that DNA sequence here: The CDS sequence is provided in
Supplemental File S13.
Go back and click on the link to the protein sequence of AS2 and copy and paste that protein sequence
here: The protein sequence is provided in Supplemental File S13.
STUDENT PRE-LABORATORY PREPARATION
Instructions to be given to students: The goals of this week’s laboratory are to determine the AS1
and AS2 PCR products sizes by gel electrophoresis and use publically available databases to gather
the wild type protein sequences of AS1 and AS2 and the cDNA sequences that encode these proteins. To
help you understand the electrophoresis process, please watch the following videos prior to attending the
laboratory. The instructor will expect that you have a general idea of how to make and load a gel and how
to analyze PCR products after gel electrophoresis.
https://www.dnalc.org/resources/animations/gelelectrophoresis.html
http://www.jove.com/video/3923/agarose-gel-electrophoresis-for-the-separation-of-dna-fragments
IN-LABORATORY DISCUSSION
I begin the in-laboratory discussion by asking the students to volunteer how gel electrophoresis works.
Based on the information they provide I add to the discussion as needed to make sure students understand
how gel electrophoresis works. In particular, I remind students of the features of DNA structure that make
gel electrophoresis possible and make sure they understand how large vs. small DNAs will migrate
through the gel. Because I still use ethidium bromide for gel electrophoresis I also discuss how ethidium
bromide works for the visualization of DNA and also warn the students the ethidium bromide is a
hazardous material.
Even if the students seem to understand the basics of gel electrophoresis, I usually have a practice gel
available so I can demonstrate how to load a gel to small groups of students. I also allow students to load
the remaining wells of the practice gel and give them the opportunity to ask questions before we begin
loading the real gel. Finally, I clarify the order in which the gel will be loaded. I like to load the AS1 PCR
products amplified from wild type, as1, and as2 plants first, and then have the students load the AS2
PCR products amplified from wild type, as1 and as2 plants.
SAMPLE STUDENT HANDOUT
Introduction: During this week’s laboratory activities, the AS1 and AS2 PCR products that were
amplified from wild type, as1, and as2 plants will be analyzed by gel electrophoresis. During the gel
electrophoresis, you will query databases to retrieve the wild type protein sequences of AS1 and AS2 and
the cDNA sequences that encode these proteins. Once the protein sequences of AS1 and AS2 have been
retrieved from the databases, the instructor will demonstrate how to submit these sequences to a software
program that will make a three-dimensional (3-D) model of the AS1 and AS2 proteins.
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: Prepare the samples
The PCR reactions from last week have a total volume of 25 L. Pipette 10 L of this reaction into a
clean 500 L tube. Place the remaining 15 L of PCR product on ice for the instructor to collect and
refreeze. To the 10 L of PCR, you need to add 6X loading buffer. Determine how much loading buffer
you need to add to the PCR so that the amount of the loading buffer you add is 1/6 of the total volume
(PCR and loading buffer combined).
Amount of 6X loading buffer that needs to be added:_______________
Once the instructor has checked your amount, add the 6X loading buffer to the PCR. Briefly, spin the
PCR.
Task 2: Load the gel
The instructor will load the molecular weight marker. When the instructor calls your group, load your
entire sample into the well. Indicate on the gel sign-up sheet which lane your sample was loaded.
When the samples are loaded, the instructor will start the gel. The gel will run at 6V/cm for 60-90
minutes.
Task 3: Bioinformatics
Go to the TAIR (The Arabidopsis Information Resource) website at www.arabidopsis.org. Under “Search
genes”, enter “AS1” into the “Gene Name, starts with” search box then click “submit query”. Click on
AT2G37630 and a new page will open. Read over the details on the page and make sure you are viewing
the information for the AS1 gene in Arabidopsis. Look closely at the “Map Detail Image.” This image
depicts the AS1 gene location on the chromosome. In this images, thick lines are exons and thin lines are
introns. Thick dark blue lines are the protein coding region of the gene. How many exons are in the AS1
gene? How many introns? Is the protein coding sequence interrupted by introns?
Scroll down the page and until you find the “Sequence” heading. Click on the link to the cDNA sequence
of AS1 and copy and paste that DNA sequence here:
Go back and click on the link to the AS1 CDS sequence (the coding region of the gene from the start to
stop codon). Copy and paste that DNA sequence here:
Go back and click on the link to the protein sequence of AS1 and copy and paste that protein sequence
here:
Compare the sequence of the AS1 CDS with the AS1 cDNA sequence. In the AS1 cDNA sequence,
underline the start and stop codons. One easy way to do this is to use the “Search” feature in Word and
search the cDNA sequence for the first and last few bases of the CDS sequence.
Repeat the process to find the AS2 sequences. Note that, because the AS2 gene is alternatively spliced,
there are multiple possible splice variants, i.e. mRNA products (AT1G65620.1, AT1G65620.2,
AT1G65620.3, and AT1G65620.4). For each of the four splice variants, how many exons are present?
How many introns? Is the protein coding sequence interrupted by introns?
AT1G65620.1:
AT1G65620.2:
AT1G65620.3:
AT1G65620.4:
Click on the link to the cDNA sequence of AS2 and copy and paste that DNA sequence here:
Go back and click on the link to the CDS sequence (the coding region of the gene from start to stop
codon) of AS2 and copy and paste that DNA sequence here:
Go back and click on the link to the protein sequence of AS2 and copy and paste that protein sequence
here:
Compare the sequence of the AS1 CDS with the AS2 cDNA sequence. In the AS2 cDNA sequence,
underline the start and stop codons. One easy way to do this is to use the “Search” feature in Word and
search the cDNA sequence for the first and last few bases of the CDS sequence.
Task 4: Submit protein sequences to I-TASSER
The instructor will demonstrate to the entire class how to submit amino acid sequences to I-TASSER.
Please look over the I-TASSER website: http://zhanglab.ccmb.med.umich.edu/I-TASSER/ and review the
background information http://zhanglab.ccmb.med.umich.edu/I-TASSER/about.html to gain a basic
understanding of what this program does. Write a short paragraph in your own words that summarizes
what I-TASSER will do with the amino acid sequences of AS1 and AS2.
Task 5: Analyze gel
The instructor will send you an electronic copy of the gel that was started at the beginning of the
laboratory. In addition, the instructor will send you a description of the DNA molecular weight standard
loaded onto the gel. Compare the known sizes of the DNA fragments in the standard with the AS1 and
AS2 PCR products. Answer the following:
1. Did your group’s PCR “work?” If yes, explain how you know. If no, explain what may have gone
wrong.
2. What is the approximate size of your group’s PCR product? Remember that during last week’s
laboratory, you predicted the sizes of the AS1 and AS2 PCR product. Does the size of your group’s PCR
product correspond to the predictions you made last week? If the answer to this last question is no,
provide a possible explanation.