LABORATORY 4: MAKING SURE YOU
HAVE CREATED A RECOMBINANT
PLASMID
LSSI Alum,
Lindsey Engle,
Granite Hills High School
El Cajon, CA
What have we already done?
In your own words, in your lab notebook, briefly
write down what we accomplished in Lab 2. (You
have 2 minutes)
• We used restriction enzymes (BamHI and HindIII)
to “digest” or cut the pKAN-R plasmid and pARA
plasmid.
Why did we do this?
• To remove the rFP gene and its promoter
• To open the pARA plasmid so we can insert the
rFP gene
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Restriction analysis of pKAN-R and pARA
pKAN-R
BamHI
5,512 bp
PBAD-rfp
806 bp
pARA
4,872 bp
376 bp
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In your own words, in your lab notebook, briefly
write down what we accomplished in lab 3. (2
minutes)
• We used DNA ligase to form a recombinant
plasmid called pARA-R. **If we were successful!
Why did we do this?
• So that we can give this plasmid to E. coli to
express the red fluorescent protein gene through
transformation!
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Recombinant construct of interest
How will we know if we were
successful?
1. We must confirm the digestion of the pKAN-R
and pARA plasmids using a control for
comparison.
2. We must confirm the ligation has successfully
created the pARA-R plasmid.
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Gel Electrophoresis for DNA
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In your lab notebook write down the
answers to these pre-lab questions,
and discuss your answers with your
partner.
1. Why do DNA restriction fragments and plasmids
separate when analyzed by gel electrophoresis?
2. Why is it important to identify and verify a
recombinant plasmid?
3. When DNA fragments are joined with a DNA
ligase an array of products is created. How does
this happen?
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Before we begin, let’s review some
terms. . .
• Vocabulary Activity
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Chapter 4: Making Sure You’ve Got
What You Need
• Learning Goals
– Describe the importance of verifying products
created in the genetic engineering process
– Predict the relative speed of DNA fragments
and plasmids in gel electrophoresis
– Separate and identify DNA restriction
fragments and plasmids using gel
electrophoresis
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Chapter 4: Suggested Activity Sequence
• Session 1: Active reading and class
discussion pp 69-74
• Session 2: Lab 4 “Verification of
Restriction and Digestion Using Gel
Electrophoresis” pp 75-79
• Session 3: Analyze gel pictures; Ch. 4
questions p 80
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Lab Prep & Aliquoting Guidelines
Reagents/Supplies
140 ul LD/class Loading Dye (LD)
The (LD) contains the gel green stain please see note before using
Aliquot
12ul/group
Storage Temp
RT
130 ul M/class (M)
12ul/group
RT
The Marker (M) is ready to use – please
see note before using
6 L of 1x SB buffer/kit
RT
10 gels per class. If you prefer to have
N/A
4o
your students pour their own gels, please
let us know when you make your
reservation on LARS.
Equipment/Supplies
10 Student boxes with the following:
1 p20 micropipette
1 microfuge rack
1 p200 micropipette
1 bag of microfuge tubes
1 p1000 micropipette
1 waste and 1 ice bucket
1 box of refillable tips (2 ul-200 ul)
4 Mini centrifuges
10 Gel Electrophoresis chambers
10 Gel trays
10 Gel combs
5 Power supplies
1 Embi Tec/PrepOne System
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Notes
Protect from light keep in amber tubes until ready to
use.
Invert or finger vortex tube to ensure solution is
mixed before aliquoting
Protect from light keep in amber tubes
Invert or finger vortex tube to ensure solution is
mixed before aliquoting
Please return bottles
Please return any unused gels.
Discard used gels in the trash.
Safety
General Lab Safety Guidelines
• Use laboratory coats, safety glasses and gloves as appropriate.
• Avoid restrictive clothing and open-toed shoes.
• No eating or drinking in the lab.
• Make sure that students are familiar with the operating instructions and safety precautions
before they use any of the lab equipment.
• Check all MSDS (Material Safety Data Sheets) for all chemicals and reagents in the lab before
preparing and running the lab.
• Wash hands at the conclusion of the lab.
Lab Safety Guidelines for lab 4
• Follow specific safety and disposal guidelines for the DNA gel green DNA stain or the DNA stain
you are using.
• Use caution with hot liquids and glassware. Wear heat-proof gloves to move hot glassware and
liquids.
• Make sure the power is turned off on power supplies before connecting electrodes.
• After gel run is complete, turn off power supply then unplug electrodes.
• If the power is on, do not touch the buffer or electrophoresis equipment as you may receive an
electrical shock.
• Never leave the electrophoresis power unit on without supervision. There is a risk of fire if the
buffer leaks out or if the buffer should evaporate completely during electrophoresis.
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Lab 4: Verification of Restriction and
Digestion Using Gel Electrophoresis
Method Overview:
– Use gel electrophoresis to separate and
visualize DNA fragments from Lab 2 and
recombinant plasmids from Lab 3
– Analyze sizes of fragments and plasmids
using DNA ladder (marker) and control
lanes
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Student Workflow
• Prepare samples (see Table 4.1)
• Load and run gels (40-50 min.)
– Students load samples in same order
• Remove gels and photograph
(teacher)
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Table 4.1: Addition of reagents to the geK- , geK+, geA- , geA+, and geLJG tubes
Sequence
Step 4 and 5 Distilled water (dH2O)
Step6
Load in g dye (LD)
-
geK-
geK+
geA-
geA+
geLIG
tube
tube
tu be
tube
tube
4 µL
4 µL
4 µL
4 µL
3 µL
2 µL
2µl
2 µL
2 µL
2 µL
-
Step 6 Nondigested pKAN -R (K- }
Step 7 Digested p KAN-R (K+)
Step 7 Nondigested pARA (A- )
Step 7 Digested pARA (A+)
Step 8 Ligated p l a s m i d (L IG)
4 µL
4 µL
4 µL
4 µL
5 µL
Running Agarose Gel
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Follow the lab protocol/methods in
the student guide
• The Loading Dye (LD) contains the DNA stainGel Green.
• Students should follow the procedure in the
table on page 77 to prepare samples for
loading into gel wells.
• The DNA Marker/Ladder (M) has been
prepared and is ready to use.
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Photographing Gels
• When gel run is complete (approximately 15-20
minutes) a good way to determine if the gels have run
long enough is to observe the orange G (OG) which will
be visible on the gel while it is running.
• Once the orange G is ~ 1/3 of the way down the gel,
the gels are ready to be photographed.
• Plan system to track gels by period and group number
• Use the PrepOne system to visualize and photograph
the gels.
• Allow students to use cell phones to capture images of
the gels or teacher can capture images using his/her
phone.
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Making a Hypothesis
• Based on your knowledge of labs 2 and 3,
draw your gel, label each lane with what was
added, and predict the resulting bands that
will be seen if we are successful with the
digestion and ligation. Refer to the
background information in the student guide.
• Label your diagram “Hypothesis.”
• Here is a sample diagram. . .
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Lab 4 Results
• Draw your gel electrophoresis results in the
same way as your hypothesis.
Hint: Your students can take pictures of the
results, print and paste in their lab reports.
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• Compare your hypothesis and results.
• Was the digestion successful?
– What is your evidence?
• Was the ligation successful?
– What is your evidence?
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Restriction analysis of pKAN-R and pARA
Restriction fragments after digest with Hind III and BamH I
BamH I
Hind III
4,706 bp
BamH I
Hind III
4,496 bp
Hind III
BamH I
806 bp
Hind III
BamH I
376 bp
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M
K-
K+
A-
A+
Lig Dye
(Smaller fragment not observed)
Analysis
What are some possible explanations for your
results if they were different than you
predicted?
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Plasmid Size and Shape Will
Influence Migration
Through Gel
• Many possible constructs from ligation
– Any complimentary sticky ends may
recombine
• Plasmids shapes may differ:
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Lab 4 Conclusions
• The original plasmids without the enzyme are present. The
multiple bands in the K- and A- columns result from various
conformations of the plasmid. The plasmid (due to handling and
replication) may have several possible forms such as supercoiled,
open-circle or multimer.
• The digest was successful. The bands in K+ column (at 4705 bp and
807 bp) and the band in the A+ column (at 4495 bp) shows the
plasmids were digested. The band of the digested pARA at 377 bp
was not observed but the presence of the larger fragment shows
the plasmid was digested.
• Presence of pARA-R confirms that the ligation has been successful.
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DNA Separation Lab with Betty Burkhard
• Shows what it’s like to do these labs in a
classroom setting
• Talks about the importance of gel
electrophoresis training for students
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