This document provides instructions for restriction ligation cloning using restriction enzymes and ligation. It describes isolating a DNA target from a source vector using restriction enzyme digestion or PCR. It also describes digesting a cloning vector with restriction enzymes and ligating the DNA target into the vector. The resulting ligation product is transformed into bacteria and colonies are selected for using antibiotic resistance. Positive clones are isolated and verified by restriction digestion and sequencing. The document provides two examples - one where the DNA target is isolated using restriction enzyme digestion, and another where PCR is used to isolate the target. It aims to guide the user through the basic steps of restriction ligation cloning.

1. DON’T CLONE ALONE
Learn about
Restriction Ligation Cloning
In 2 easy steps

2. Step 1
A short guide for the
Restriction Ligation Cloning Method

3. 3
Step 1: A Short Guide for the Restriction Ligation Cloning Method
CLONING
A technique for reproducing DNA fragments
This traditional cloning method permits the entry of a DNA fragment of interest
into a vector to be amplified

4. 4
Step 1: A short guide for the Restriction Ligation Cloning Method
The Cloning Strategy Overview
1. DNA target isolation by
a. Option 1: Restriction Enzyme digest
b. Option 2: PCR
2. Restriction Enzyme digest of a cloning vector
3. Ligation of the DNA of interest and the cloning vector
4. Transformation with the ligation products
5. Growth on agar plates with selection for antibiotic resistance
6. Isolation of desired DNA clone
7. Verification of the cloning process

5. 5
Step 1: A short guide for the Restriction Ligation Cloning Method
The Cloning Strategy Overview
1. DNA target isolation by
a. Option 1: Restriction Enzyme digest
b. Option 2: PCR
2. Restriction Enzyme digest of a cloning vector
3. Ligation of the DNA of interest and the cloning vector
4. Transformation with the ligation products
5. Growth on agar plates with selection for antibiotic resistance
6. Isolation of desired DNA clone
7. Verification of the cloning process

6. 6
Restriction Enzymes
Cut DNA at a specific recognition site and generate one of two types of
ends:
• Blunt ends: In which both DNA strands terminate in a base pair.
Blunt ends are always compatible with each other:
• Sticky ends (long overhangs): Contains a stretch of unpaired nucleotides at the
end of a DNA molecule. These unpaired nucleotides can be on either strand,
creating either:
• 5' overhangs:
• 3' overhangs:
1a) DNA target isolation by Restriction Enzyme Digest

7. 7
1a) DNA target isolation by Restriction Enzyme Digest
Restriction Enzymes
Search for the presence of unique restriction sites in the source DNA that
can be targeted with restriction enzymes to isolate the DNA target.

8. 8
Step 1: A short guide for the Restriction Ligation Cloning Method
The Cloning Strategy Overview
1. DNA target isolation by
a. Option 1: Restriction Enzyme digest
b. Option 2: PCR
2. Restriction Enzyme digest of a cloning vector
3. Ligation of the DNA of interest and the cloning vector
4. Transformation with the ligation products
5. Growth on agar plates with selection for antibiotic resistance
6. Isolation of desired DNA clone
7. Verification of the cloning process

9. 9
1b) DNA target isolation by PCR
PCR
• If no appropriate restriction sites can be found in the source DNA, then PCR
may be chosen as the method of isolation of the DNA target.
• To excise the DNA target, a primer pair will be designed.
• 5’ restriction sites, which are compatible to the region of insertion in the
cloning vector, may be added as primer overhang sequence.

10. 10
Step 1: A short guide for the Restriction Ligation Cloning Method
The Cloning Strategy Overview
1. DNA target isolation by
a. Option 1: Restriction Enzyme digest
b. Option 2: PCR
2. Restriction Enzyme digest of a cloning vector
3. Ligation of the DNA of interest and the cloning vector
4. Transformation with the ligation products
5. Growth on agar plates with selection for antibiotic resistance
6. Isolation of desired DNA clone
7. Verification of the cloning process

11. 11
2) Restriction Enzyme Digest of a Cloning Vector
Cloning Vector:
A DNA molecule that is used to transport cloned sequences between a test
tube and a biological host
The Vector Properties:
A. Can promote autonomous replication inside a host
B. Contain a genetic marker for selection
C. Has unique restriction sites to facilitate cloning of DNA target
D. Has a minimum amount of non-essential DNA to optimize cloning

12. 12
2) Restriction Enzyme Digest of a Cloning Vector
A host vector is cut with the same restriction enzymes (or different ones,
yielding the same sticky ends)
• The source DNA and the vector plasmid can be digested with a single restriction enzyme for
non-directional cloning, or with 2 different restriction enzymes for directional cloning
• Using two different enzymes that create blunt ends will also create a non-directional cloning
strategy, and for any non-directional strategies, additional screening may be required to
determine the desired placement of the insert in the vector

13. 13
(End Modification)
Avoid the possibility of self-ligation:
• Just before the ligation step, the vector can be dephosphorylated by Alkaline Phosphatase in
order to lower the chances of self ligation. In this process, the 5’ phosphate group, which the
ligase needs for phosphodiester bond formation, is removed.
• Depending on how the insert and vector are prepared, other ‘end’ treatment may be required
i.e. blunting, A-tailing and phosphorylation

14. 14
Step 1: A short guide for the Restriction Ligation Cloning Method
The Cloning Strategy Overview
1. DNA target isolation by
a. Option 1: Restriction Enzyme digest
b. Option 2: PCR
2. Restriction Enzyme digest of a cloning vector
3. Ligation of the DNA of interest and the cloning vector
4. Transformation with the ligation products
5. Growth on agar plates with selection for antibiotic resistance
6. Isolation of desired DNA clone
7. Verification of the cloning process

15. 15
3) Ligation of DNA Sample Products and Cloning Vector
The gene is inserted into the plasmid and ligated with Ligase in a process
called – DNA Recombination
• A Ligase is selected to covalently join the phosphate backbone of DNA with blunt or
compatible cohesive ends
• The DNA ends have to collide and stay together for the Ligase to join them

16. 16
Step 1: A short guide for the Restriction Ligation Cloning Method
The Cloning Strategy Overview
1. DNA target isolation by
a. Option 1: Restriction Enzyme digest
b. Option 2: PCR
2. Restriction Enzyme digest of a cloning vector
3. Ligation of the DNA of interest and the cloning vector
4. Transformation with the ligation products
5. Growth on agar plates with selection for antibiotic resistance
6. Isolation of desired DNA clone
7. Verification of the cloning process

17. 17
4) Transformation with the Ligation Products
The new ligation product
is inserted into Bacteria
host cells (usually E. coli)

18. 18
Step 1: A short guide for the Restriction Ligation Cloning Method
The Cloning Strategy Overview
1. DNA target isolation by
a. Option 1: Restriction Enzyme digest
b. Option 2: PCR
2. Restriction Enzyme digest of a cloning vector
3. Ligation of the DNA of interest and the cloning vector
4. Transformation with the ligation products
5. Growth on agar plates with selection for antibiotic resistance
6. Isolation of desired DNA clone
7. Verification of the cloning process

19. 19
5) Growth
Selective amplification:
…on agar plates (or any other suitable media) with selection for antibiotic resistance:
• A specific antibiotic is added to the agar plates to kill the host without any protection. The
transformed host is protected by the antibiotic resistance gene
• The growth of colonies will therefore demonstrate an antibiotic resistance and the presence of
the newly acquired insert (the gene of interest)

20. 20
Step 1: A short guide for the Restriction Ligation Cloning Method
The Cloning Strategy Overview
1. DNA target isolation by
a. Option 1: Restriction Enzyme digest
b. Option 2: PCR
2. Restriction Enzyme digest of a cloning vector
3. Ligation of the DNA of interest and the cloning vector
4. Transformation with the ligation products
5. Growth on agar plates with selection for antibiotic resistance
6. Isolation of desired DNA clone
7. Verification of the cloning process

21. 21
6) Isolation
Identification of colonies that contains the product
The identification of host colonies with the desired product can be done
in 3 ways:
1. Colony PCR – either with the primers used for amplification of the target, or preferably by
primers based in the vector on both sides of the target
2. Restriction enzyme digestion based screening – digesting host DNA to identify positive
colonies
3. Plasmid DNA migration-based screening – comparison of plasmid size to identify the
required size product

22. 22
Step 1: A short guide for the Restriction Ligation Cloning Method
The Cloning Strategy Overview
1. DNA target isolation by
a. Option 1: Restriction Enzyme digest
b. Option 2: PCR
2. Restriction Enzyme digest of a cloning vector
3. Ligation of the DNA of interest and the cloning vector
4. Transformation with the ligation products
5. Growth on agar plates with selection for antibiotic resistance
6. Isolation of desired DNA clone
7. Verification of the cloning process

23. 23
7) Verification: Was the Cloning Process Successful?
Make sure that the DNA target (the insert) was inserted successfully to the
backbone and the host is expressing this insert (the gene)
Common verification methods:
• Restriction Digest followed by Gel Electrophoresis - Separates restriction
enzyme digested DNA fragments by molecular weight
• DNA Sequencing - Provides complete order of bases in a DNA fragment. You will
want to compare the sequence to your template sequence from the design phase,
by performing a pairwise alignment

24. Step 2
Practice makes the design perfect…

25. 25
A short guide for the Restriction Ligation Cloning Method
The Cloning Strategy Overview – let’s not forget the steps!
1. DNA target isolation by
a. Option 1: Restriction Enzyme digest
b. Option 2: PCR
2. Restriction Enzyme digest of a cloning vector
3. Ligation of the DNA of interest and the cloning vector
4. Transformation with the ligation products
5. Growth on agar plates with selection for antibiotic resistance
6. Isolation of desired DNA clone
7. Verification of the cloning process
The exercises will focus on steps 1, 2, 3 and 7 – the design and verification
phases in Genome Compiler!

26. 26
By the end of this practice you will:
Understand the cloning process by using:
• Restriction Enzymes to excise the insert
• PCR to excise the insert
Learn how to verify the cloning process by:
• Sequence alignment
• Virtual digest
Step 2: Practice makes the design perfect…

27. 27
Open a Genome Compiler account in order to start your practice:
http://www.genomecompiler.com
You may want to know first:
• Sign up will take only a few seconds
• You can decide if you want to use it online or use the downloadable version.
• Genome Compiler is FREE for academia users
Practice Makes the Design Perfect

28. 28
Look for these materials inside Sample Projects folder
in the Materials Box:
• Source vector: PUC57 + Gene A (the insert)
• The Backbone: pcDNA3.1 C-HA
We’ll also use these restriction enzymes:
Xbal, Notl and EagI
The Materials for the Exercise

29. 29
Exercise Overview
Using Genome Compiler’s Restriction Ligation wizard, we will ligate Gene A from source vector pUC57
into the MCS of backbone vector pcDNA3.1 C-HA by option (1) “Digest” Insert and then by option (2)
“PCR” insert and check it is in frame with the downstream HA tag on the backbone
HA tag

30. 30
OPTION 1
GENERATE THE INSERT BY DIGEST

31. 31
1. Digest PUC57 + Gene A with: XbaI and EagI
2. Linearize backbone pcDNA3 by digest with: XbaI and NotI
3. Generate product
4. Check Gene A is cloned into MCS of pcDNA3 in frame to downstream HA tag using the
ORF detection tool
5. Save and finish to generate a new cloned product and save the cloning wizard steps
Let’s begin!!!...
Option 1 Overview: Generate the Insert by Digest

32. 32
Open the Restriction Ligation Cloning
Wizard
Put Some Magic in Your Cloning Project

33. 33
Select the ‘Insert’ tab
Drag and drop the
source vector pUC57 +
Gene A from the
Sample Projects
folder inside the
Materials Box on the
left into the right
panel
1) DNA target isolation by restriction digest

34. 34
On the left, choose to
generate the insert by
Digest
1) DNA target isolation by restriction digest

35. 35
In the cut with boxes, type the restriction enzymes XbaI and
then EagI.
These enzymes are good candidates because they don’t cut the
insert sequence:
Question
Why not choose NdeI? This restriction site is also present.
Click on it and see why you can’t use it
1) DNA target isolation by restriction digest

36. 36
Select the insert
fragment from the
table (1165bps)
As you click on each
fragment you will see it
selected on the right -
on the plasmid
You can also see the
sticky ends on the
cloning minimap
1) DNA target isolation by restriction digest

37. 37
2) Restriction Enzyme digest of a cloning vector
Select the green
Backbone tab along
the top
Drag and drop the
Backbone
pcDNA3.1 C-HA
from the Sample
Projects folder in the
Materials Box to the
right panel

38. 38
We want to insert our
Gene A into the MCS
We need to zoom into
the sequence to look
at the cut sites in this
region
Click on the MCS
region
Select the sequence
tab along the top
2) Restriction Enzyme digest of a cloning vector

39. 39
Right click on an enzyme
restriction site in the sequence view
Select ‘Show only single cutters’
From here we can see the single
cutters in the MCS which can help us
decide which enzymes to use.
2) Restriction Enzyme digest of a cloning vector

40. 40
For the first cut site,
from the cut with
box, select XbaI
This restriction site is
the same as on the
insert. It can also be
seen in the MCS as a
single cutter.
Look on the cloning
experiment summary
down the bottom to
view the restriction sites
2) Restriction Enzyme digest of a cloning vector

41. 41
Question:
For the second cut site,
ideally we would also
choose EagI as this is what
we will excise the insert
with
Why can’t we? Select it
and see why.
From the second cut
with box, choose NotI
instead of EagI
2) Restriction Enzyme digest of a cloning vector
Question:
How come we can use NotI?

42. 42
Note back:
If the restriction sites are not compatible
then you can always modify the ends…
(out of scope for this practice)
(End modification)

43. 43
Select the backbone fragment from this table (5425bp)
2) Restriction Enzyme digest of a cloning vector

44. 44
Select the product
tab.
Congratulations, you
have a product!
Check that your
sequence is in frame
to the downstream HA
tag. Click on the
“ORF detection”
tool.
3) Ligation of the DNA of Interest and the Plasmid Vector

45. 45
The ORF detection tool
has revealed that there is
no ORF running from the
first ATG of the insert
through to the HA tag.
So let’s use some primers
to excise the insert
instead…
3) Ligation of the DNA of Interest and the Plasmid Vector
HA tag
ORF
arrows

46. 46
OPTION 2
GENERATE THE INSERT USING PCR

47. 47
1. Excise Gene A from pUC57 using PCR
2. Linearize backbone pcDNA3.1 C-HA by digest using XbaI and NotI (we already did this
previously so keep the same settings)
3. Generate product
4. Check Gene A is cloned into MCS of pcDNA3 in frame to downstream HA tag using the
ORF detection tool
5. Save and finish to generate a new cloned product and save the cloning wizard steps
Let’s begin!!!...
Option 2 Overview: Generate the Insert Using PCR

48. 48
Select the Insert
tab and this time
choose to
Generate by PCR
Select the ‘Auto
design’ option to
design your
primers using
Primer3
1) DNA target isolation by PCR

49. 49
At this step, we will design
primers to excise Gene A
from PUC57
Select the Gene A part
in the circular view and
choose Use selection
Important – use the
arrows to de-select the
last stop codon from
the sequence selection
(this is important to
ensure transcriptional
read-through to the HA
tag)
1) DNA target isolation by PCR

50. 50
1) DNA target isolation by PCR
Press Generate primers and
select the primer pair with the
lowest Tm difference
Click Use selected primer pair

51. 51
1) DNA target isolation by PCR
The small minimap
indicates there is still no
product, and the
product tab is de-
activated.
Question
Why is there no cloned
product?
Hint: Open up the
errors panel to see
the issues

52. 52
1) DNA target isolation by PCR
We need to add
restriction sites to our
primers which are the
same/compatible with
the enzymes we chose to
digest the backbone
with.
Select the Edit primer
and add overhang
button.

53. 53
For the forward primer (Primer 1) –
add the restriction site NotI
You can also add some spacer
sequence to adjust the frame
Additionally add some leader
sequence which will be added after
the restriction site
1) DNA target isolation by PCR

54. 54
For the reverse primer (Primer 2) –
change the restriction site to XbaI
1) DNA target isolation by PCR

55. 55
1) DNA target isolation by PCR
The errors have disappeared!

56. 56
2) Restriction enzyme digest of a cloning vector
Keep the same digest
settings that we used for
the backbone as earlier
(Slides 36-42)

57. 57
3) Ligation of the DNA of Interest and the Plasmid Vector
Select the product tab.
Congratulations, you
have a product!
Check that your sequence
is in frame to the
downstream HA tag. Click
on the “ORF detection”
tool.

58. 58
3) Ligation of the DNA of Interest and the Plasmid Vector
The ORF detection tool has revealed
that there is an ORF running from
the first ATG of the insert through to
the HA tag.
Click next to move to the finalize
step!
HA tag
ORF
arrows

59. 59
Click finish to:
- Generate the
cloned product as
a new project
- Save the cloning
wizard steps
Congratulations, You have a cloned product in frame!

60. 60
In order to verify that the cloning process was successful, we can use
one of these options:
1. Run a virtual digest - to see the expected fragment sizes using
Genome Compiler’s virtual digest tool
Learn More
2. Run a pairwise alignment - to verify the sequence using the Clustal
Omega algorithm in Genome Compiler
Learn more
The Verification Phase

61. Learn more about Genome Compiler
www.genomecompiler.com