Experiment 7: Genetic Engineering

Cloning is the process of isolating a segment of DNA (usually a gene) from an organism and making multiple exact
copies of the DNA by replication in a host organism (such as bacteria). DNA can be isolated by restriction enzyme
digestion of genomic DNA or amplified using PCR. The segment of DNA is then inserted into in a small circular
DNA, known as a vector or plasmid. The new combination of DNA is known as recombinant DNA. Cloning allows
the production of large amounts of pure DNA, which can then be used in applications such as sequencing, creating
transgenic (genetically modified) plants and animals, and the production of purified proteins on a large scale. The
techniques to create and clone recombinant DNA, and the applications where recombinant DNA is used are
collectively known as genetic engineering. This lab will demonstrate the process of inserting a fragment of DNA
produced by PCR into a cloning vector and transforming that vector into bacteria.
Cloning using PCR
PCR is a quick and powerful technique to amplify a fragment of DNA in a short amount of time. Within just a few
hours, trillions of copies of a fragment of DNA can be produced. However, PCR has some limitations. First, PCR
requires sequence-specific primers in order to guide DNA polymerase to the segment of DNA to be amplified.
Creating these primers therefore requires that sequence information about the gene of interest is known. Second,
the fact that PCR is a very sensitive technology also presents a limitation. The right conditions have to be achieved
in order for the primers to anneal to the correct segment of DNA to avoid off-target amplification.
Designing primers for PCR
Primers are one of the most important parts of any PCR reaction, since they determine whether the proper
segment of DNA will be amplified or not. Primers are short (18-30 nucleotide long), single-stranded pieces of DNA,
also known as oligonucleotides, which anneal at each end of the target sequence to be amplified. Currently,
companies specialize in the production of primers for use in PCR, but it is the responsibility of the researcher to
determine the sequence that the company should produce. For traditional PCR, two primers are designed, each
one flanking the target sequence to be amplified (Figure 7-1).

Figure 7-1: Primers are designed to be complementary to the sequence flanking either end of a gene of interest.

In traditional PCR, sequence information of the target gene is used to design primers which are perfectly
complementary to the target sequence (Figure 7-1). However, when sequence information of the target gene is
not known, the approach to designing primers can be modified.
Amplifying DNA of unknown sequence by PCR
Despite the limitations mentioned previously, PCR can be used to amplify DNA without the exact sequence
information for a particular gene of interest. One way to approach the problem is to use sequence data for the
gene of interest in related species that have already been cloned and sequenced. Especially when the sequences
of many related species are known, highly conserved regions of the gene sequence can be used to design primers
that will likely amplify the gene from the genomic DNA of the new species. An alternative approach is possible if
the protein sequence of the gene of interest is known. Starting with the protein sequence, it is possible to work
back to the possible DNA sequence using the genetic code. One drawback to this approach is that because of the
degenerate nature of the genetic code, there is more than one possible DNA sequence that would result in the
known amino acid sequence.
The gene that will be studied in this lab encodes for the enzyme glyceraldehyde-3-phosphate dehydrogenase
(GAPDH). GAPDH is a critical enzyme involved in both glycolysis (Figure 7-2) and in the Calvin cycle (dark reactions
that produce carbohydrates) in plants. Due to its vital role in these basic processes, the GAPDH protein is highly
conserved between organisms, particularly in the active site domains of the protein. This conservation between
species allows us to create a collection of primers that should anneal with some specificity to the GAPDH gene in
any species of plant.

Figure 7-2: The process of glycolysis. GAPDH catalyses the conversion of glyceraldehyde-3-phosphate into 1,3
bisphosphoglycerate.
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Creating primers for PCR of DNA of unknown sequence

To clone the GAPDH gene from a variety of species of plants, primers need to be designed that are complimentary
to conserved regions of the gene. Since even conserved regions of the gene will not be identical between species,
a best guess of the gene sequence is made by comparing the alignment of GAPDH genes from many related
organisms (figure 7-3). Although not all the bases are conserved, the differences at each base position between
species is not random. The fourth base of each sequence in Figure 7-3 is either a T or an A. The base that is more
commonly found at each position can be used for designing the primer, in this case, a T would be used. Using this
information, a set of primers will be designed, each containing the conserved bases at positions where there is
little to no variability. The positions that are variable, such as position 3 (denoted as B) will have a different base
at that position in each primer in the set, in this case a G, T or C. This mixture of primers is known as a set of
degenerate primers.

Figure 7-3: Designing PCR primers by deriving a consensus sequence and introducing degeneracy. The GAPDH
gene is duplicated in some organisms, and therefore has more than one sequence, each with a slightly different
name and acronym.
Another method for creating degenerate primers is to use amino acid sequence information for a protein. The
degeneracy of the genetic code (figure 7-4) means that for one amino acid sequence, there are multiple possible
DNA sequences that could produce the same amino acid sequence. So, in order to reduce the number of
degenerate primers needed for PCR, a region of the protein with amino acids that are encoded by only 1 or 2
codons each can be selected.

Figure 7-4: The genetic code - amino acids and the DNA codons for each.

Nested PCR
By creating degenerate primers that will not necessarily have exact sequence complementarity to our target gene
of interest, the problem arises that DNA other than our desired gene will be amplified (i.e. off-target
amplification). In order to address this issue and increase the chance that we are amplifying the GAPDH gene, we
will perform nested PCR (figure 7-5). In nested PCR, two rounds of PCR are performed sequentially. The first round
uses degenerate primers. This PCR reaction is then treated with exonuclease to remove the first set of primers.
The second PCR reaction uses the first PCR products as a template with a new set of primers which anneal to
regions inside of the target region of the first PCR reaction. The primary advantage of nested PCR is that if the first
set of primers amplified DNA outside of the target DNA, it is unlikely that the second set of primers will do the
same. The most prominent fragment of DNA amplified should be our gene of interest.
We will use the plant genomic DNA you isolated from various fruits and vegetables during the GMO lab to attempt
to clone the GAPDH gene from each sample. As a control for the nested PCR for GAPDH, we will be using genomic
DNA from Arabidopsis thaliana, a member of the mustard family and the most extensively studied model plant.
Arabidopsis is an ideal model plant due to its small genome which has been fully sequenced (125 Mb) and its rapid
life cycle of only 6 weeks. Arabidopsis contains a family of 4 different GAPDH genes (figure 7-3). The primer
mixtures we will be using amplify the same region in all these genes and give products that are all approximately
1 kb in size (see figure 7-6).

Figure 7-5: Nested PCR process

1 2 3 4 5 6 7 8 9

1 2 3 4 5 6 7 8 9

Figure 7-6: Examples of possible results with two different plants.

(Sample types for lanes 1-3 and 6-9 are the same for both gels above)
Template (PCR reaction)
Lane 1: Molecular weight marker
Lane 2: Arabidopsis genomic DNA (initial PCR)
Lane 3: Arabidopsis genomic DNA (nested PCR)
Lane 4: Genomic DNA of test plant (initial PCR)
Lane 5: Genomic DNA of test plant (nested PCR)
Lane 6: Plasmid DNA with cloned GAPDH gene (initial PCR)
Lane 7: Plasmid DNA with cloned GAPDH gene (nested PCR)
Lane 8: Negative control - water (initial PCR)
Lane 9: Negative control - water (nested PCR)

Ligation and transformation of PCR products into a plasmid vector

Once we have amplified our gene of interest, the next step in cloning is to insert the DNA into a plasmid. Plasmids
are small circular pieces of DNA found in bacteria and in some eukaryotes such as yeast. The advantage of inserting
our DNA into a plasmid is that the bacterium can do the work of replicating the DNA. Replication of plasmids
occurs independent of the timing of the replication of the bacterial chromosome. Depending on the type of
plasmid, a single bacterium can produce hundreds of copies (up to 700!) of the same plasmid. By growing large
numbers of the bacteria in culture, many more copies of the DNA are produced than through the process of PCR.
6

This large amount of DNA can be purified to analyze further (such as for sequencing the gene) or to manipulate
further (such as during the production of GMO crops).
The plasmid we will insert our PCR product into is the pJet1.2 blunted vector (figure 7-7). The vector has several
features that make it ideal for cloning. First, it is a blunted vector, which means we can insert our PCR products
into the vector with minimal preparation of the PCR product. Second, the plasmid contains the eco47IR gene. This
gene codes for the Eco47I restriction enzyme, which when expressed, is toxic to E. coli cells. The eco47IR gene is
positioned in the plasmid so that if our PCR product is successfully inserted into the plasmid, the eco47IR gene will
be disrupted and the enzyme will no longer be expressed (see figure 7-7). This allows us to easily select for
transformed bacteria that contain not only the plasmid, but a plasmid that contains our PCR product.

Figure 7-7: pJet1.2 blunted vector

We will use the process of ligation to insert our PCR product into the pJet1.2 plasmid. Ligation is the process of
joining two pieces of linear DNA into a single piece. DNA ligase (the enzyme that catalyzes the last step in DNA
replication) is used to join the two pieces of DNA together. The most commonly used ligase comes from the
bacteriophage T4 and is named T4 DNA ligase. As with all DNA ligases, this enzyme catalyzes the formation of a
phosphodiester bond between the 3-OH of one piece to DNA and the 5-phosphate on the second piece of DNA.
Immediately following ligation, the ligated plasmid is then transformed into bacteria.

Exercise 7: Genetic engineering

LEARNING OBJECTIVES
Describe the basic steps of cloning
Compare and contrast simple and nested PCR
Compare the transformation efficiency of an intact plasmid to a ligated plasmid.
PROCEDURES
Plant genomic DNA for this lab will come from the test food DNA isolated in section 5.1 of Experiment 5:
Genetically modified organisms. For the PCR reactions listed here, the test food must be a fresh vegetable or
plant of some kind; processed foods will not have adequate DNA to ensure proper amplification of the target
gene, GAPDH.
7.1 Setting up initial PCR reactions
The students at each table will set up PCR reactions as a group. On your ice, you should have three tubes for the
control DNA templates: one is the negative control and contains sterile water, the second tube contains
Arabidopsis genomic DNA, and the third is a positive control containing the pGAP plasmid DNA. As a group, you
will also set up one experimental sample, using the DNA you isolated from one test food you brought in last
week.
1. Obtain the genomic DNA from your test food from last week, thaw the sample in your hand, mix it gently
by flicking, and spin at 6,000xg for 5 minutes to pellet the InstaGene matrix. Place the tube on ice with
the other DNA template tubes.
2. Label the 4 PCR tubes at your table with the numbers 1-4 and initial them on the top and side so that they
can be identified next week. Remember, labels on the top of the tube are likely to be lost due to the
heated lid of the PCR machine. Keep the tubes on ice for the next series of steps. Fill in table 7-1 as each
step is completed.
3. Flick to thoroughly mix the contents of the 2x blue mastermix with initial primers (2x MMIP) tube and
centrifuge 10 sec to force contents to the bottom of the tube.
4. Pipet 20 l of the MMIP into each PCR tube on ice.
Caution: Be sure not to disturb the InstaGene pellet while transferring the test food DNA sample to the
PCR reaction.
5. Using a fresh tip for each sample, add 20 l of the appropriate DNA template to each tube using table 71 as a guide. Gently pipet up and down to mix reagents. Recap tubes.

Table 7-1. Initial PCR reaction components (blue)

Tube
label

Sample name

Blue Master
Mix

DNA Template

Total
Volume

Negative control
(sterile water)

20 l

20 l water

40 l

Arabidopsis gDNA

3
4

Positive control pGAP

plasmid
Plant (test food)
gDNA

6. Leave your samples on ice until your TA asks you to bring your samples to the thermal cycler for loading.
For organizational purposes, your TA will place your tubes in numerical order and in straight rows inside
the thermal cycler according to the chart provided. Sign the sheet next to the thermal cycler indicating
the position of your groups tubes.
7. When all the samples are in the machine, the PCR program will be run. Finished samples will be stored in
the freezer until the next lab.
8. Clean your station by disposing of ice in the sink.

7.1 Section review

1. What is the purpose of using degenerate primers for this initial PCR reaction?
2. Why is the GAPDH gene a good candidate gene to attempt to amplify from a variety of different plants?

7.2 Setting up nested PCR reactions

Again, as a group, you will prepare 4 PCR samples. One nested PCR will be performed for each gDNA sample
amplified in the initial round of PCR (initial PCR samples 2 and 4 only). In addition, you will perform one positive
control using new control pGAP plasmid DNA and one negative control with sterile water instead of DNA template.
1. Retrieve initial PCR samples 2 and 4 that you prepared previously. Leave samples 1 and 3 in the freezer to
be analyzed on a gel next week. Use Table 7-1 to determine the contents of each PCR tube. Samples 2 and
4 should be the Arabidopsis gDNA and test food PCR samples, respectively. Place tubes 2 and 4 on ice.
2. PCR reactions 2 and 4 contain genomic DNA, PCR product and any unincorporated primers from the initial
PCR reaction. In order for the nested PCR primers to work optimally, we must remove the unincorporated
initial primers using exonuclease I. Using a fresh tip each time, pipet 1 ul of exonuclease I into each blue
initial PCR reaction (2 and 4).

3. Incubate the exonuclease reactions at 37C for 15 minutes.

4. Heat inactivate the exonuclease by incubating at 80C for 15 minutes.
5. Label 2 microcentrifuge tubes at your table for the exonuclease-treated PCR reactions as follows: exo A
for the exonuclease treated PCR from Arabidopsis gDNA and exo T for the exonuclease treated PCR from
the test food DNA. Initial the tubes on the top and side as well.
6. Add 98 l of sterile water to each microcentrifuge tubes you just labelled.
7. Using a fresh tip each time, add 2 l of each exonuclease-treated PCR reaction into the appropriate
microcentrifuge tube. This step will dilute the initial PCR reactions to the correct concentration for the
nested PCR reaction. Place these tubes with the diluted DNA templates on ice.
Caution: Return the tubes labelled 2 and 4 to the freezer with tubes 1 and 3 to analyze on the agarose gel next
week.
8. Vortex or flick the tubes labelled exo A and exo T to mix. Centrifuge 10 sec to force contents to the
bottom of the tube.
9. Label the 4 PCR tubes at your table with the numbers 5-8 and initial them on the top and side so that they
can be identified next week. Keep the tubes on ice for the next series of steps. Fill in table 7-2 as each step
is completed.
10. Flick to thoroughly mix the contents of the 2x yellow mastermix with nested primers (2x MMNP) tube and
centrifuge 10 sec to force contents to the bottom of the tube.
11. Pipet 20 l of the MMNP into each PCR tube on ice.
12. Using a fresh tip for each sample, add 20 l of the appropriate DNA template to each tube using table 72 as a guide. Gently pipet up and down to mix reagents. Recap tubes.
13. Leave your samples on ice until your TA asks you to bring your samples to the thermal cycler for loading.
For organizational purposes, your TA will place your tubes in numerical order and in straight rows inside
the thermal cycler according to the chart provided. Sign the sheet next to the thermal cycler indicating
the position of your groups tubes.
14. When all the samples are in the machine, the PCR program will be run. Finished samples will be stored in
the freezer until the next lab.
15. Clean your station by disposing of ice in the sink.

10

Table 7-2. Nested PCR reaction components (yellow)

Tube
label
5
6
7
8

Sample (template)
name
Negative control
(sterile water)
Exonuclease-treated
Arabidopsis gDNA
initial PCR
Positive control pGAP
plasmid
Exonuclease-treated
Plant (test food)
gDNA initial PCR

Yellow
Master Mix

DNA Template

Total
Volume

20 l

20 l water

40 l

7.2 Section review

1. What is the possible outcome for the nested PCR reaction if the exonuclease were not inactivated?
2. What is the benefit of conducting two rounds of PCR in this lab?

7.3 Agarose gel electrophoresis of DNA

This part of the lab will be completed in groups of four at each table. Each group of students will prepare one
agarose gel for separating the control and experimental PCR products for both the initial and nested PCR
reactions by electrophoresis.
At your station, you will find a small microtube rack that contains your Orange G loading dye (LD), the
molecular weight ruler (MWR), 4 PCR tubes, and eight microtubes with snipped lids. This microtubes are
necessary when using the centrifuge. The loading dye co-migrates with the DNA and allows us to track the
progress of the gel as it is running. The MWR contains DNA pieces of the following sizes to serve as reference
bands: 500 bp, 1000 bp, 1500 bp, 2000 bp, 2500 bp, 3000 bp, 4000 bp, 4500 bp, 5000 bp.
Note: To review the entire procedure for pouring, running and staining an agarose gel, see Sections 3.4-3.6.
1. Prepare a gel using 75 ml of a 1% agarose gel solution. Calculate the amount of agarose needed to make
this solution. (Remember 1% = 1g per 100 ml.) Check your answer with your TA to ensure your calculation
is correct.
Grams of agarose needed = ____________
Warning: Be careful around hot plates and hot liquids. Always wear protective gloves near heat sources or when
handling hot liquids. Be careful not to get burned.

11

2. Retrieve the PCR samples you prepared previously. Use table 7-1 and 7-2 to determine the contents of
each small tube. If you prepared your PCRs in the correct order, the numbered lid indicates the
components of each tube.
3. Place PCR tubes into adapter tubes and pulse-spin for about 3 seconds to bring all the liquid to the bottom.
Make sure the machine is balanced and the rotor lid is well secured.
4. Keep your samples on ice during the preparation for electrophoresis.
Note: Samples 1-4 and 5-8 will be treated differently. Pay careful attention during the next few steps.
5. Prepare samples 5-8 first. Since the nested PCR products (tubes 5-8) will also be used for ligation into the
pJet1.2 plasmid, we must aliquot a small amount of each sample into new tubes to combine with loading
dye (LD) for gel electrophoresis. Label the PCR tubes in your microtube rack with the following numbers:
5D, 6D, 7D, 8D. The D mark will indicate these are the tubes to add loading dye to.
6. Add 10 l of loading dye (LD) to each of the tubes in the microtube rack (5D, 6D, 7D, 8D).
7. Using a fresh tip for each sample, pipet 10 l of the appropriate PCR sample (5-8 only!) into the
appropriate D tube containing loading dye. Gently pipet up and down to mix reagents.

Important!!: For samples 5-8, keep the remainder of the PCR reaction on ice for purification in a later step.
8. Now prepare samples 1-4. Using a fresh tip for each addition, add 10 l of Orange G loading dye (LD)
directly to each PCR sample. Gently pipet up and down to mix reagents.
9. Load 20 l of samples 1-8, one sample per lane. It will be easiest to make comparisons if the initial and
nested PCR samples of each DNA template are loaded next to each other (see figure 7-6). Keep track of
the order of the samples on the gel. Write the PCR tube number, the type of DNA and whether the sample
is of the initial or nested PCR reactions in Table 7-3 by the lane in which the samples were loaded. Each
team member should load at least one sample. There are a few extra lanes to use in case of mistakes!
Remember to leave room to also load 20 l of the MWR.

12

Table 7-3. Gel loading record

Lane number
1
2
3
4
5
6
7
8
9
10
11
12
13
14

Sample loaded
Dont load anything here!

Dont load anything here!

10. Run the gel at 150V for 30-45 minutes.

11. Turn off the power supply. Unplug the electrode leads.
12. Remove the cover from the gel apparatus.
Caution: This gel is a lower percentage of agarose than we have been using so far in the labs. The gel will be
more fragile than previous gels. Use care during any handling steps.
13. Designate one group member to handle the gel. That person must always wear gloves while handling the
gel, the staining tray and the staining solution.
14. Stain and destain the gel as in Experiment 3, Section 3.6. Remember that a longer destain gives easier to
distinguish bands. Sketch your results now, but gels may be left in water with gentle agitation to analyze
again at the end of lab.
15. Sketch your results in Table 7-4.

13

Table 7-4. Initial and nested PCR results.

1 2 3 4 5 6 7 8 9 10 11 12 13 14

7.3 Section review

1. Compare and contrast the results you obtained for the Arabidopsis gDNA template and the test food
template. Were similar products (in terms of size and number of bands) obtained for these two templates?
2. Compare and contrast the results you obtained from the initial PCR reactions compared to the nested PCR
reactions.

7.4 Purification of nested PCR products (yellow)

Before proceeding to ligate our PCR products into the pJet1.2 plasmid vector, smaller impurities, such as
unicorporated primers or dNTPs and Taq polymerase must be removed.
This part of the lab will be completed in groups of four at each table. Each group of students will purify the PCR
products of only the Arabidopsis and test food nested PCR reactions (PCR tubes labeled 6 and 8, see table 7-2.)
At your station, you will find a small microtube rack that contains 2 each of PCR Kleen spin columns, 2.0 ml wash
tubes, and 1.5 ml collection tubes.
1. Locate the tubes labelled 6 and 8 from your PCR tubes on ice. Use table 7-2 to determine the contents
of each small tube. If you prepared your PCRs in the correct order, the numbered lid indicates the
components of each tube.
2. Place PCR tubes into adapter tubes and pulse-spin for about 3 seconds to bring all the liquid to the
bottom. Make sure the machine is balanced and the rotor lid is well secured.
14

3. Keep your samples on ice during the preparation for purification.

4. Label the 2 spin columns at your table with the numbers 6 and 8 and initial them on the side so that
they can be identified.
5. Label the 2.0 ml wash tubes at your table with the numbers 6W and 8W and initial them on the top and
side so that they can be identified. The W indicates these are the wash tubes.
6. Label the 1.5 ml collection tubes at your table with the numbers 6P and 8P and initial them on the top
and side so that they can be identified next week. The P indicates these tubes will contain your purified
PCR products.
7. Resuspend the resin in the spin column by vortexing for 5 seconds.
8. Remove the cap, snap off the tip and place each column in a 2.0 ml wash tube (labeled 6W and 8W).
9. Centrifuge the columns in wash tubes for 1 minute in the microcentrifuge at 735 x g (~2800 rpm). The
TA will help coordinate the loading of the microcentrifuge at this step.
10. Retrieve your spin columns in wash tubes and remove the wash tube. Place the spin columns in the
appropriate 1.5ml collection tubes (labeled 6P and 8P).
11. Apply the full 30 l of each PCR reaction to the top center of the column bed in the appropriate column.
Be careful not to disturb the column bed. See figure 7-8 below.

Figure 7-8. Applying PCR sample to spin column.

12. Centrifuge the columns in 1.5 ml collection tubes for 2 minutes in the microcentrifuge at 735 x g (~2800
rpm). The TA will help coordinate the loading of the microcentrifuge at this step.
13. Save the purified samples which are in the bottom of the 1.5 ml collection tubes (6P and 8P). These
samples will be used for the ligation reaction in the next section. Your teaching assistant will store the
samples in the freezer until the next lab.

15

7.5 Ligation of nested PCR products into pJet1.2 vector

The first reaction for this procedure will remove the 3 nucleotide overhang from the purified PCR products you
prepared previously (6P and 8P) using the proofreading polymerase. Once the PCR product is blunted, the second
reaction will ligate the PCR products into the pJet1.2 vector.
This part of the lab will be completed in groups of four at each table. On your ice, you will find ligation reaction
buffer (LB, contains the proofreading polymerase) and at your station on a microtube rack, you will find 3 PCR
tubes. The T4 DNA ligase (T4) and the pJet1.2 blunted vector (V) are on ice and will be supplied by the teaching
assistant when you are ready to use them.
Note: There are steps in this part of the procedure which require pipetting very small amounts of liquid (between
.5 l and 1 l). Remember that this is a very small amount, and that it may feel like a very small movement of the
plunger when you take up the liquid. Take a look at the pipet tip to ensure that you have pipetted the correct
volume of reagent. Have a group member that is confident with pipetting complete this part of the procedure to
conserve reagents.
1. Retrieve the purified PCR samples (6P and 8P) that you prepared previously.
2. Label the three PCR tubes 6, 8 and NC for negative control (this reaction will contain only vector) and
initial them on the top and side so that they can be identified. Keep the tubes on ice for the next series of
steps. Fill in table 7-5 as each step is completed. You will add ligation buffer and PCR product first and
incubate for the proofreading reaction (steps 6-9), then you will add the vector and ligase (step 10).
3. Centrifuge the tube containing ligation reaction buffer (LB) for 10 seconds to force contents to the bottom
of the tube.
4. Pipet 8 l of the ligation reaction buffer (LB, contains polymerase) into each PCR tube on ice.
5. Using a fresh tip for each sample, add 1 l of the appropriate PCR product to each tube using table 7-5 as
a guide. The negative control already has 1 l of water added for you. Each reaction now contains 9 l.
Gently pipet up and down to mix reagents. Recap tubes.
Table 7-5. Ligation reaction components.
Tube
label

Sample name

Ligation
buffer

PCR product

pJet1.2
vector

T4 DNA
ligase

Final
Volume

NC

Negative control
(vector only)

8 l

1 l water

.5 l

.5 l

10 l

Arabidopsis gDNA

Plant (test food)

gDNA

6. Centrifuge in the microcentrifuge for 10 seconds to force contents to the bottom of the tube.

16

7. Place the tube at 70C for 5 minutes.

8. Place the tube on ice to cool for 2 minutes.
9. Centrifuge in the microcentrifuge for 10 seconds to force contents to the bottom of the tube. Place the
tube at room temperature.
10. Using the same tubes containing the proofreading reactions you prepared in steps 6-9, and a fresh tip for
each sample, add .5 l each of the pJet1.2 vector and T4 DNA ligase to each reaction. Each reaction now
contains 10 l. Gently pipet up and down to mix reagents. Recap tubes.
11. Centrifuge in the microcentrifuge for 10 seconds to force contents to the bottom of the tube.
12. Incubate the ligation reactions at room temperature for 10 minutes.
13. Place the ligation reactions on ice until ready to proceed with the transformation step (section 7.6).

7.6 Transformation of ligation reactions

Note: Depending on your lab section, this transformation procedure will be performed differently. The TA will
let you know which procedure to proceed with.
7.6a If you will be performing the transformation as we did for the pGLO plasmid, use the procedure below:
1. Label the four pink filled microtubes on ice NC for negative control, 6, 8 and PC for positive control (pGLO
plasmid) and initial them on the top and side so that they can be identified. Keep the tubes on ice for the next
series of steps. Fill in table 7-6a as each step is completed.
Table 7-6a. Transformation reaction components.
Tube/plate
label

Sample name

Ligation
reaction

Calcium
chloride
solution

Final
Volume

NC

Negative control
(vector only)

5 l

250 l

255 l

Arabidopsis gDNA

250 l

Plant (test food)

gDNA

250 l

PC

pGLO
(positive control)

1 l

250 l

2. Label the yellow microtube LB (nutrient broth). It already contains the nutrient broth needed for the
recovery stage. Keep this tube on the microtube rack.

17

3. Following sterile technique, use a new inoculation loop to pick up 2-3 single colonies of bacteria from the
starter plate. You do not need more since each colony contains millions of E. coli cells.
4. Immerse the loop into the transformation solution in the NC tube. Spin the loop between your index finger
and thumb until the colony is dispersed. There should be no floating chunks.
5. Place the tube back on ice and repeat steps 3 and 4 for the remaining tubes of transformation solution (6,
8 and PC).
6. Using a fresh tip for each sample, pipet 5 l of the appropriate ligation reaction into tubes NC, 6 and 8.
Pipet 1 l of the pGLO plasmid DNA into the tube labeled PC. Keep the tubes on ice.
7. Incubate all 4 tubes on ice for 10 minutes. Be sure the bottoms of the tubes are in contact with the ice.
8. During the incubation period, label your plates. Retrieve the 4 LB Amp IPTG plates and label each on the
bottom of the plate using table 7-6a as a guide. Be sure to also write the names/initials of the members
of your group and the lab day and time on each of the plates.
9. Heat shock the cell suspensions. Place the foam holder with your tubes into a water bath or heat block
set at 42C for exactly 50 seconds. If using a water bath be sure the bottom of each tube is in contact with
the water.
10. Immediately after the 50-second heat shock period, place the tubes back on ice.
11. Incubate the tubes on ice for 2 minutes.
12. Take your foam holder with the 4 tubes back to your lab bench. Using sterile technique, pipet 250 l of LB
broth (from the yellow tube) into the NC tube. With a new sterile pipet tip, repeat this step for the other
3 pink tubes.
13. Incubate all 4 tubes at room temperature for 10 minutes.
14. Retrieve the labelled LB Amp IPTG plates and pipet the entire volume of the appropriate transformation
reaction onto the appropriate plate using table 7-6a as a guide.
15. Using a fresh inoculation loop for each sample, very gently spread the bacteria around the plate. Do not
spread for more than 10 seconds. Immediately close the plate and wrap the plates in parafilm. Place the
plates in a stack in the incubator. Your teaching assistant will coordinate arranging the plates.
16. The plates will be incubated at 37C for several days, and then transferred to cold storage until next week.

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7.6b If you will be performing the new transformation method, use the procedure below:
The goal of this part of the lab is to transform bacterial cells using the ligation reactions you prepared in section
7.5. As a positive control for the transformation procedure, you will also be transforming bacterial cells with the
pGLO plasmid.
This part of the lab will be completed in groups of four at each table. On your ice, you will find 1 tube of competent
cells. At your station, you will find 4 microtubes on the microtube rack. In the incubator, you will find 4 LB Amp
IPTG plates.
1. Retrieve the 4 LB Amp IPTG plates and label each on the bottom of the plate using table 7-6b as a guide.
Be sure to also write the names/initials of the members of your group and the lab day and time on each
of the plates. Return these plates in the incubator until they are needed.
2. Label the four microtubes from the microtube rack NC for negative control, 6, 8 and PC for positive control
(pGLO plasmid) and initial them on the top and side so that they can be identified. Keep the tubes on ice
for the next series of steps. Fill in table 7-6b as each step is completed.
3. Retrieve the ligation reactions that you prepared previously.

Table 7-6b. Transformation reaction components.

Tube/plate
label

Sample name

Ligation
reaction

Competent
cells

Final
Volume

NC

Negative control
(vector only)

5 l

50 l

55 l

Arabidopsis gDNA

Plant (test food)

gDNA

PC

pGLO
(positive control)

1 l

4. Using a fresh tip for each sample, pipet 5 l of the appropriate ligation reaction into tubes NC, 6 and 8.
Pipet 1 l of the pGLO plasmid DNA into the tube labeled PC. Keep the tubes on ice.
5. Using a fresh tip for each sample, very gently pipet the competent cells up and down and then pipet 50 l
of cells into each tube using table 7-6b as a guide. Very gently pipet up and down the contents of each
tube as you add cells, and then return the tubes to the ice.
6. Incubate the transformation reactions on ice for 10 minutes.
7. Retrieve the warm LB Amp IPTG plates from the incubator and pipet the entire volume of the appropriate
transformation reaction onto the appropriate plates using table 7-6b as a guide.
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8. Using a fresh inoculation loop for each sample, very gently spread the bacteria around the plate. Do not
spread for more than 10 seconds. Immediately close the plate and wrap the plates in parafilm. Place the
plates in a stack in the incubator. Your teaching assistant will coordinate arranging the plates.
9. The plates will be incubated at 37C for several days, and then transferred to cold storage until next week.

7.7 Analysis of transformation results

1. Before looking at your plates, predict the answers to the question below for each of the four plates. Enter
your predictions in table 7-7.
a. Do you expect to see no growth, colonies or a lawn?
2. Obtain your groups set of plates. Unwrap them as much as necessary to make your observations. You
should strive to keep the plates closed.
3. For each plate, record the answers to the following in Table 7-7.
a. Does each plate have no growth, discrete colonies or a lawn?
b. If there is colony growth, count the number. A great number of colonies can be estimated as
you did in section 6.2.
Note: It is possible that satellite colonies may grow using the ligation method in this protocol. Count only the
large colonies, not the tiny colonies which surround larger colonies.

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Table 7-7. Transformation reaction results.

Plate
label

Predicted growth

Growth
Colonies, lawn or
Colony
no growth
count/estimate

NC

PC

7.7 Section review

1. Should you expect to see colonies on the negative control (NC) plate? Recalling the components of this
ligation reaction, explain your prediction for this plate. Did your results match the prediction?
2. Compare plates 6 and 8 with plate PC (positive control with the intact pGLO plasmid). How is transformation
efficiency affected by ligation?
3. Compare plates 6 and 8. Which plate gave more colonies? Provide a few explanations for any differences
in the number of colonies on these plates.

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