Molecular cloning can be sometimes tricky with significant challenges involved. Overcome the challenges with the essential knowledge and tips for successful cloning.

1. Guide to molecular cloning
Essential knowledge and tips for successful clonings
Introduction
1. Bacterial plasmids are circular molecules of double-stranded
DNA, ranging from 1 to 200 kb in size.
2. Plasmid vectors are used to replicate or express the gene or
DNA sequence of interest. This gene or sequence is inserted
into the multiple cloning site.
3. Antibiotic resistance genes are used as selectable markers.
Bacteria carrying a plasmid vector with such a gene survive
in antibiotic cell culture, while bacteria that did not take up
the plasmid vector die.
4. Insert DNA is part of a gene or DNA sequence that is
inserted into a larger DNA vector using a recombinant
DNA technique, such as ligation. This allows it to be
multiplied, selected, further manipulated, or expressed in a
host organism.
5. The lacZ gene is needed for blue-white screening, which
shows the successfully transformed bacteria. lacZ is within
the multiple cloning site
Step 1 — Preparing the plasmid vector and insert DNA
A — PCR and cleanup
Insert DNA is generated from the PCR products.
• Amplify the insert DNA from the template using PCR.
• Refine the PCR products via DNA cleanup with the QIAquick®
PCR Purification Kit.
B — Restriction digestion
Treat the plasmid vector and insert DNA with restriction digestion enzymes to generate the desired
binding site for downstream applications.
Step 2 — Running the preparative agarose gel
To separate the prepared vector and insert bands from unwanted material, such as
incompletely digested DNA, enzymes, and other small molecules, run a preparative agarose
gel. Then perform gel extraction cleanup with the QIAquick Gel Extraction Kit.
Step 3 — Ligation
Mix the insert DNA and vector DNA at appropriate concentrations and incubate for
1–24 h at 15°C.
Step 4 — Transformation
Transformation is where the bacteria (usually Escherichia coli) take up plasmid vectors from
the environment. The two typical transformation methods are heat shock and electroporation.
Heat shock uses CaCl2
treatment, chilling and mixing with the plasmid vector on ice, and
then 90 s to 2 min heat shock at 42°C.
Electroporation uses micro- to millisecond electric pulses to temporarily disrupt cell membrane
semipermeability, giving increased plasmid vector uptake. Cell pretreatment with ice-cold
10% glycerol is needed.
Step 5 — Blue-white screening
Blue-white screening involves plating transformed cells on media containing antibiotics,
IPTG, and X-Gal.
The beta galactosidase gene (lacZ-gene) is cloned within the multiple cloning site and
repressed by the lacZ repressor. In an intact vector (without insert DNA), the galactosidase
enzyme is active, and in the presence of IPTG, the lacZ repressor is removed, meaning the
enzyme can cleave X-Gal (provided on the plates). This cleavage generates a blue color.
If the gene is destroyed by the insert, the enzyme cannot be expressed and the colony
remains white.
Select the white colonies. Incubate them overnight with the appropriate antibiotics at 37°C.
Extract plasmid DNA using the QIAquick Gel Extraction Kit or QIAprep®
Spin Miniprep Kit
on the QIAvac®
. The DNA yield and quality depend on the quality of cell lysate used for
the purification.
Sample to Insight
Tip
Estimate the plasmid DNA
concentration before doing digestions.
Run a DNA agarose gel to analyze
fragments between 0.1 and 25 kb, or
use a NanoDrop®
Spectrophotometer
for rapid and easy assessment of
concentration.desired binding site for
downstream applications.
Tips
1. The restriction enzyme should be
kept on ice and added last.
2. Thorough mixing is extremely
important.
3. The restriction enzyme should not
exceed more than 10% of the total
reaction volume.
4. For large numbers of digests, make
a reaction master mix consisting
of water, buffer, and enzyme, and
aliquot this into tubes containing
the DNA to be digested.
Tips
Increase your recovery rate by:
1. Using Buffer TAE instead of
Buffer TBE
2. Applying sufficient agitation during
the dissolution step
3. Reducing the centrifugation speed
during the binding step
4. Extending the incubation time of
the elution buffer to 5 min
Know-how
Watch the demonstration at
www.qiagen.com/Cleanup-Protocol/
Tips
1. Ligating fragments with 4-bp 3’
or 5’ overhanging ends requires
far less ligase than more complex
or blunt-end ligations. Blunt-end
ligations require 10–100 times
more enzyme than sticky-end
ligations to achieve the same
efficiency.
2.High-quality DNA also requires
less ligase.
3. Ligate sticky ends at 12–15°C to
maintain the balance between
annealing of the ends (difficult
at higher temperatures) and the
activity of the enzyme (diminished
at lower temperatures).
4. Perform blunt-end ligations at room
temperature, but not above 30°C.
5. Use two parallel ligations (one
without insert DNA, one without
vector DNA) as negative controls
to check for background clones
arising from self-ligation of
inefficiently phosphatized vector.
Tips
Check transformation efficiency.
Transform competent cells with 1 ng
of a control plasmid containing an
antibiotic resistance gene. Plate onto
LB-agar plates containing the relevant
antibiotic(s). Compare the number
of colonies obtained with the control
plasmid to the number obtained with
the plasmid of interest to compare
transformation efficiency.
Know-how
1. Blue: plasmid vector without
insert DNA
2. White: plasmid vector with
insert DNA
3. Use the QuickLyse Miniprep Kit
to get your answer in just 9 min.
Tips
1. Resuspend bacteria completely
to maximize the number of cells
exposed to the lysis reagents.
2. For large-scale purification of
low-copy plasmids, increase the
buffer volumes. This increases the
efficiency of alkaline lysis.
3. Avoid vigorous stirring or
vortexing as this can shear the
bacterial chromosome, which will
then copurify with the plasmid
DNA. Mix the solution gently but
thoroughly by inverting the lysis
vessel 4–6 times.
4. Lysis should not proceed for longer
than 5 min, to avoid irreversible
plasmid denaturation.
Know-how
Read more about selecting suitable
restriction enzymes at
www.qiagen.com/Restriction-Enzymes.
Learn more about molecular cloning at www.qiagen.com/Knowledge-and-Support
For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and
user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor.
Trademarks: QIAGEN®
, QIAprep®
, QIAquick®
, QIAvac®
(QIAGEN Group); NanoDrop®
(ThermoScientific)
1100661 01/2016 © QIAGEN, all rights reserved
The last step is a sequence check to check that the insert is not carrying any mutations. It uses
primers located in the vector to proof the insertion sites.
Bacteria
Plasmids Bacterial
chromosome
Plasmid vector
Insert DNA
The origin
of replication
Selectable markers
(e.g., antibiotic resistance genes)
VacuumQIAquick and MinElute Procedure
QIAquick and MinElute Procedure
PCR or other
enzymatic reaction or
solubilized gel slice
Pure DNA fragment
From solutions 5 min
From gels 15 min
ProcedurePCR or other
enzymatic reaction
orsolubilized gel slice
QIAcube
Pure DNA fragment
From solutions 5 min
From gels 15 min
Vacuum
Vacuum
lacZ gene
lacZ geneA multiple cloning site (polylinker)
with many restriction enzyme sites
Blunt-ended DNA fragments Sticky-ended DNA fragments
Plasmids PCR Product
A T T
T
T T
T
GT A A
A
A A
A
CG G GC C C
C
T A AA ACA T TT TGC C C
G
AAT TC
T CG
A GC
G G G
G
AG C
TC G
Heat shock transformation Electroporation transformation
42°
0
40
Untransformed bacteria unchanged plasmids
Transformed
bacteria
Transformed bacteria
Low throughput 1–24 minipreps
Overnight cultures
Resuspend
Lyse
Neutralize
Clear
lysates
QIAprep QIAprep
Vacuum
Vacuum
Bind
Wash
Elute
High-purity
plasmid DNA
12 minipreps – 26 min
24 minipreps – 30 min
12 minipreps – 20 min
24 minipreps – 25 min
High-purity
plasmid DNA
QIAprep Spin
in microcentrifuges on vacuum manifolds
Low throughput 1–24 minipreps
AGT CG C
TCA GC G
Phosphodiester
bonds
DNA ligation
DNA ligase