A Seminar Discussion on PCR 

and Cloning of Insert
Did anybody said 
“CLONING”?

By,
Atul Kakrana
atulkakrana@yahoo.com

An abandoned presentation for SURE SHOT 
5/24/2010
CLONING ‐ Atul Kakrana
 Prologue

• A 1971 paper in the Journal of Molecular Biology by Kleppe and Nobel laureate H. Gobind Khorana first 
described a method using an enzymatic assay to replicate a short DNA template with primers in vitro
• This early manifestation of the basic PCR principle did not receive much attention, and the invention of the 
polymerase chain reaction in 1983 is generally credited to Kary Mullis.
• Mullis received the Nobel prize in chemistry (1993) for his development of the Polymerase Chain 
Reaction (PCR)

PCR BASED CLONING

• Cloning PCR products into plasmid vectors is a common downstream application of PCR.
• Commonly used strategy for PCR cloning is to add restriction enzyme recognition sites to the ends of PCR 
primers . The PCR product is then digested and cloned into the desired vector.

An abandoned presentation for SURE SHOT 
5/24/2010
CLONING ‐ Atul Kakrana
 PCR – Nostrum for Science and Research

• The polymerase chain reaction (PCR) is a technique to amplify a single or few copies of a piece of DNA across 
several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. 
• The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA 
melting and enzymatic replication of the DNA.

The  Program:
• Initial Heating – A step required only for heat activation in case of Hot‐start PCR. 94‐98°C /1‐9min 

• The Cycle: 
– Denaturation : It causes melting of the DNA template by disrupting the hydrogen bonds between 
complementary bases, yielding single strands of DNA.  94‐96 ° C/20‐30 Sec

– Annealing  :  It allows annealing of the primers to the single‐stranded DNA template. 55‐65 ° C/20‐40 Sec

– Polymerization :  In this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA 
template strand by adding dNTPs that are complementary to the template in 5' to 3' direction. The 
temperature depends upon the polymerase used i.e simple Taq polymerase has optimum activity at 72 ° C.

– Final Polymerization : This single step is occasionally performed after the last PCR cycle to ensure that any 
remaining single‐stranded DNA is fully extended. 70–74 °C/5–15 min.

• Hold : This is the optional most important step of the whole reaction (Gives you freedom to sleep over your PCR).

4-8° C

An abandoned presentation for SURE SHOT 
5/24/2010
CLONING ‐ Atul Kakrana
 Polymerase Chain Reaction

An abandoned presentation for SURE SHOT 
5/24/2010
CLONING ‐ Atul Kakrana
 Optimizing PCR
• Annealing Temperature : Run a Gradient PCR with increments of 2 ° C from 55 -65 ° C. If product composition is known
then annealing temperature can also be calculated with relation TaOpt = 0.3TmPrimer + 0.7TmProduct - 14.9 (W.Rychlik et al.,
1990).

• DNA Polymerase : The lack in 3' to 5' proofreading of the Taq polymerase enzyme results in a high error rate (mutations per
nucleotide per cycle) of approximately 1 in 9,000 bases, which affects the fidelity of the PCR. It is recommended to use
“High Fidelity polymerase” for cloning and sequencing needs.

• Magnesium Concentration : Magnesium is required as a co‐factor for DNA polymerase. Inadequate thawing of MgCl2 may
result in the formation of concentration gradients within the magnesium chloride solution and contribute to failed
experiments. Low Mg conc. will lead to non specific products whereas high mg concentration will inhibit DNA polymerase
activity. Optimal conc. Is 1.8‐3.6mM.

• Deoxynucleotides : Excessive amounts of dNTPs can increase the error rate of DNA polymerase and even inhibit the
reaction. An imbalance in the proportion of the four dNTPs can result in mis‐incorporation into the newly formed DNA
strand and contribute to a decrease in the fidelity of DNA polymerase

• Contamination : To avoid contamination during setting up of PCR, filter tips and hand gloves should be used. Laminar hood
can also be used to set up a PCR. Negative Control is recommended to check contamination during handling. Control
reaction is set up in the same way as the experimental PCRs, but without template DNA added, and is performed alongside
the experimental PCRs.

An abandoned presentation for SURE SHOT 
5/24/2010
CLONING ‐ Atul Kakrana
 Troubleshooting PCR
PROBLEM CAUSES SOLUTIONS

No Amplicon Incorrect annealing temperature Run a temperature gradient in 2°C increments

Primer dimers Increase temperature and/or decrease MgCl2. Check self

complementarity of primers.
Low Yield Annealing temperature not optimal Run a temperature gradient in 2°C increments

Insufficient cycles Increase amount of cycles

Extension time too short For long products (>2kb), extension time (in mins) should be
approximately equal to the number of kb in the amplicon.
Long denaturation step , inactivating  Use 2 minute denaturation time for polymerases which do not
the enzyme require a hot‐start.

Non‐Specific  Priming starting during set up Set up reaction on ice or use a hot‐start Taq polymerase

Amplification –
Multiple Products
Annealing temperature not optimal Run a temperature gradient in 2°C increments

Primers not specific Blast primers to check specificity. Redesign primers

Annealing time too long Decrease time of annealing step

An abandoned presentation for SURE SHOT 
5/24/2010
CLONING ‐ Atul Kakrana
 CONTD.

PROBLEM CAUSES SOLUTIONS

Non‐Specific Amplification – Priming starting during set up Set up reaction on ice or use a hot‐start Taq polymerase
Smeared Product
Annealing temperature not optimal Run a temperature gradient in 2°C increments

Template degraded Minimize freeze thawing of DNA. Run template
on agarose gel to check integrity.

Extension time too short For long products (>2kb), extension time (in
mins) should be approximately equal to the
number of kb in the amplicon.
Reaction Not Reproducible or  dNTPs degraded dNTPs are very susceptible to freeze thawing.
Reaction Stopped Working Replace with a fresh aliquot
Change in component Check any new components that have been
added (eg. new batch of primers)

An abandoned presentation for SURE SHOT 
5/24/2010
CLONING ‐ Atul Kakrana
 Be specific!
• The conventional Taq DNA polymerase is active at room temperature and to a lesser degree, even on ice
• When all the reaction components are put together, nonspecific primer annealing can occur due to these
low temperatures
• Non‐specifically annealed primer can then be extended by the Taq DNA polymerase, generating
nonspecific products and lowering product yields

BE  SPECIFIC WITH YOUR PRODUCTS – USE HOT START

• Hot start PCR technique utilizes specialized DNA polymerases that gets activated  only at high 
temperature
• An antibody or inhibitor is bound to DNA polymerase that denatures and dissociates at high temperature , 
restoring  DNA polymerase activity.
• This inhibits any non specific extension of annealed primers at room temperature

NEED ULITIMATE SPECIFICITY AND YIELD – TRY HOT START + TOUCHDOWN PCR

• Touchdown PCR circumvents spurious priming by starting with highest possible annealing temperature
(Just below Tm) and lowering it with subsequent set of cycles.
• Highly specific binding takes place initially at high temperature and exponential nature of PCR increases
the copy of specific sequences thereby outcompeting the non specific sequences to which primers may
bind at following lower temperatures.

An abandoned presentation for SURE SHOT 
5/24/2010
CLONING ‐ Atul Kakrana
 Primer Designing
Things to know before primer designing

• Primer Length :  18‐20 bp of length is long enough for adequate specificity. Longer primers will have high 
Tm and will give difficulties during PCR optimization.
• Primer Melting temperature : Generally  Tm should be below 65°C to avoid secondary annealing.  Primers 
with melting temperature in the range of 58‐62°C produce good results.
• GC Content : The GC content (the number of G's and C's in the primer as a percentage of the total bases) 
of primer should be 40‐60%.
• GC Clamp : The presence of G or C bases within the last five bases from the 3' end of primers (GC clamp) 
helps promote specific binding at the 3' end due to the stronger bonding of G and C bases.
• Secondary Structures :  
1. Self Dimers : A primer self‐dimer is formed by intermolecular interactions between the two (same sense) primers, 
where the primer is homologous to itself. Tolerated within ΔG of ‐5‐6 kcal/mol.
2. Cross Dimer : Primer cross dimers are formed by intermolecular interaction between sense and antisense primers, 
where they are homologous. Tolerated within ΔG of ‐5‐6 kcal/mol.
3. Hairpins : It is formed by intramolecular interaction within the primer and should be avoided. Tolerated within ΔG of ‐
2‐3 kcal/mol.
• Repeats and Runs :  Primers with long runs of a single base should generally be avoided as they can mis 
prime, ex – ACGTAAAAAAT. Runs of 4 bp are tolerable.  Similarly,  repeat is a di‐nucleotide occurring many 
times consecutively and should be avoided. Repeats of 4 di‐nucleotides are acceptable
• Cross Homology : Blast the primers with the database of organism of interest to check the specificity of 
primers.
5/24/2010
SCREENCAST FOR PRIMER DESIGNING FOLLOWS NEXT
An abandoned presentation for SURE SHOT 
CLONING ‐ Atul Kakrana
 Primer 3 : Design Primers (Screencast)

An abandoned presentation for SURE SHOT 
5/24/2010
CLONING ‐ Atul Kakrana
Inclusion of restriction sites in primers
for directional cloning
RESTRICTION SITE ANALYSIS OF INSERT AND VECTOR

• Which sites are available in vector of interests? Check Vector map

• Which sites are already present in fragment of interest? Use freely available software's such as BioEdit or
Emboss suite to find all restriction sites present in the target sequence. JUMP TO NEXT SLIDE TO CHECK

• Avoid sites that are common in both vector and insert and choose among other sites with these criteria's
in mind:
1. Enzymes for selected sites should have a compatible buffer for efficient double digestion to expedite cloning
2. Enzymes should have heat inactivation option to avoid gel elution before ligation
3. Restriction sites should be spaced apart (at least 10bp), this improves the efficiency of double digestion
4. Both the selected sites should produce sticky ends for directional cloning, one blunt and one sticky will also lead to directional cloning
5. Spacer nucleotides have to be added at 5’ end just after the restriction sites. Every restriction site demands different number of spacer
nucleotides. Minimum number of base pairs required for efficient restriction can be checked on
http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_olignucleotides.asp

• Add selected sites one in each at 5’ end of forward and reverse primers. Restriction sites should be
included in such a way that orientation of insert is in conjugation with vector.
• An Example of primer sets with restriction sites: Spacer + Res. Site + Primer
Forward    ACACGGATCCGTATTGAAGAACGTTTGCGACTG | 58.7°C|BamHI
Reverse     TAATGTCGACCCGGCGGAAGGGAATGAAG | 58.7°C|SalI

An abandoned presentation for SURE SHOT 
5/24/2010
CLONING ‐ Atul Kakrana
 Analyze your sequence for restriction sites
(Screencast)

Analyze vector for common sites

An abandoned presentation for SURE SHOT 
5/24/2010
CLONING ‐ Atul Kakrana
 Restriction of Vector and Insert
Tips for double digestion

• Keep glycerol concentration less then 5% in a reaction, to avoid this restriction enzyme conc. Should not 
be more then 1/10 of reaction volume.
• DNA preparations may have impurities which can inhibit restriction enzyme digestion activity. Some DNA 
isolation kits even use high EDTA buffers to elute the DNA. Purify your DNA either by gel elution or PCR 
purification kit.
• Set up a control digest when using restriction enzymes. Use DNA which generates known fragmentation 
patterns.

Digestion of Vector and Insert with selected (two) restriction sites

• Digest both vector and insert with same restriction sites to favor directional cloning.
• Heat inactivation (raising the temperature to 65 or 80°C for 20 minutes) is the simplest method of
stopping a reaction.
• Heat inactivation does not work for all restriction enzymes. Phenol/chloroform extraction, gel elution or
commercial kits can be used to purify the insert.

An abandoned presentation for SURE SHOT 
5/24/2010
CLONING ‐ Atul Kakrana
 Troubleshooting your digestion reaction

PROBLEM CAUSE SOLUTION

Incomplete or No  Enzyme is inactive Test enzyme on control DNA with known multiple 
digestion sites
Reaction conditions are not  Follow recommendations for double digestion, or 
optimal try a sequential digest
DNA concentration is not  Recommended conc. Is 1 µg of DNA in a 50 µl 
optimal reaction.
Excess DNA may result in incomplete cleavage
Recognition site may be too  As a general rule, add 6 bases pairs on either side 
close to the end of the DNA  of the recognition site
fragment
Unexpected Cleavage  DNA sample is contaminated Prepare a new DNA sample
pattern
Additional recognition sites are  Confirm DNA sequence
present in DNA
Star Activity Choose correct buffer/Reduce the incubation time

An abandoned presentation for SURE SHOT 
5/24/2010
CLONING ‐ Atul Kakrana
 Finally! Eligible for ligation
The mechanism of DNA ligase is to form two covalent
phosphodiester bonds between 3' hydroxyl ends of one nucleotide
with the 5' phosphate end of another. which can be catalyzed by
two different ligases: E. coli DNA ligase and bacteriophage T4 DNA
ligase. The latter is the preferred enzyme because it can also join
blunt‐ended DNA fragments.

Tips for efficient ligation

• Before ligation, completely inactivate restriction enzyme by heat

inactivation, spin column or Phenol/EtOH purification
• Keep total DNA concentration between 1‐10 µg/ml i.e 10‐
100ng/10µl reaction mix
• Insert: Vector molar ratios between 2:1 and 6:1 are optimal for An example of ligation (sticky ends)
single insertions
• Quantify your restricted vector and insert before ligation step to
save hard labor. If you are unsure of your DNA concentration,
perform multiple ligations with varying ratios
• Ligase buffer contains ATP, which is unstable and degraded by
multiple freeze/thaw cycles. Make 10‐20ul aliquots from the
original
• Do not heat inactivate ligase if there is PEG in the reaction
buffer because transformation will be inhibited. Few
Commercially available kits contains PEG
An abandoned presentation for SURE SHOT 
5/24/2010
CLONING ‐ Atul Kakrana
 A yummy ligation recipe
Ingredients : Quantified vector and insert  (restricted), T4 Ligase , Buffer and Nuclease free water

• Select/ Choose your molar ratio,  generally Vector: Insert ratio of 1:3 is preferred
• Use this formula to calculate amount of vector and insert required for selected molar ratio:
Insert mass (ng) = 6 X [insert length  (bp)/vector length (bp)] X Vector mass (ng) 
• Alternatively online ligation calculators can also be used  to calculate vector and insert amount:
– http://www.promega.com/biomath/calc06.htm (Promega)
– http://www.fermentas.com/reviewer/app?page=Calculator&service=external&sp=Sligations (Fermentas)
– http://www.insilico.uni‐duesseldorf.de/Lig_Input.html (Heinrich‐Heine‐Universität)
• Set up your ligation mix for a total volume of 5‐10µl. This volume is enough for successful transformation 
and saves your ingredients too.

Incubation: Incubate overnight at temperature recommended by the enzyme manufacturer. More units of 
ligase can be added to shorten the time of incubation. Increase in ligation temperature also shortens the 
time of incubation required.

SCREEN CAST FOR LIGATION CALCULATION FOLLOWS

An abandoned presentation for SURE SHOT 
5/24/2010
CLONING ‐ Atul Kakrana
 Calculate your ligation mix
(Screencast)

An abandoned presentation for SURE SHOT 
5/24/2010
CLONING ‐ Atul Kakrana
 Troubleshooting : Ligation
PROBLEM CAUSE SOLUTION

No Ligation at all Insert/vector are degraded prior to or  Check DNA on Gel

during ligation
Faulty restriction
Component of ligation is missing or not
working
Over digestion of Vector and insert (Star
activity)
Hidden restriction site
The ligase was inactive
Decreased ligation  Insufficient DNA either vector or insert
efficiency
high salt or EDTA in the reaction
Transform cut and uncut vector and 
look for colonies
Always put positive control
Use of excess of restriction enzyme and 
prolonged incubation should be 
avoided
Analyze target sequence for  all 
restriction sites
Test on lambda HindIII or other 
convenient substrate
Increase insert/vector ratio
Clean up the DNA

An abandoned presentation for SURE SHOT 
5/24/2010
CLONING ‐ Atul Kakrana
 Transformation
Transformation is the genetic alteration of a cell resulting from the uptake, genomic incorporation, and expression of
environmental genetic material (DNA). Transformation occurs most commonly in bacteria, both naturally and artificially, and refers
to DNA taken up from the environment through their cell wall.

Calcium Chloride transformation
• Calcium chloride transformation is a method of promoting competence. Chilling cells in the presence
of divalent cations such as Ca2+ (in CaCl2) prepares the cell membrane to become permeable to plasmid DNA
• Cells are incubated on ice with the DNA and then briefly heat shocked (e.g. 42 °C for 30–90 seconds), which causes the
plasmid DNA to enter the cell

Tips for efficient transformation using chemical competent cells

• Competent cells are best thawed on ice and DNA should be added as soon as last bit of ice in the tube disappears
• Incubate DNA with competent cells for 30 min before heat shock. Expect 2 fold loss in transformation efficiency for every 10 
min you shorten this step
• Outgrowth medium should be SOC, it gives 2 fold higher efficiency then LB medium and incubation with shaking increases 
the efficiency to 2‐fold when compared to incubation without shaking

Electroporation

• Electroporation is another way to make holes in bacterial (and other) cells, by briefly shocking them with an electric field of
10‐20kV/cm
• Plasmid DNA can enter the cell through these holes. Natural membrane‐repair mechanisms will rapidly close these holes
after the shock. . This method is amenable to use with large plasmid DNA.

An abandoned presentation for SURE SHOT 
5/24/2010
CLONING ‐ Atul Kakrana
Troubleshooting:
Cloning
There could be several factors for unsuccessful 
cloning. This makes it imperative to set up 
controls at each step:
1. Amplification
• +Ctrl : Any working primer set with 
same DNA
• ‐Ctrl : H2O instead of DNA
2. Restriction :
• +Ctrl:  Known segment with same 
sites
• ‐Ctrl : H2O instead of water
3. Ligation :
• +Ctrl : Most of the ligation kits have 
Cut vector as control. Alternatively 
Lambda /Hind III sample cant be 
used.
4. Transformation :
• +Ctrl : Uncut Vector (Circular)
• ‐Ctrl : Cut Vector (Linear)

An abandoned presentation for SURE SHOT 
5/24/2010
CLONING ‐ Atul Kakrana
 EXCEPTION TO THE RULE OF THUMB
Does  increasing Ta improves specificity of primer always? No, not always

• It is well known that with increase in annealing temperature for PCR favors highly specific binding
between primers at template of maximum complementarities.
• But, this may not be true with every primer set. In the gel snap (Gradient PCR) below with increase in
Ta, specificity increases in primer1 but it decreases in case of primer 2 and 3.
• “Non‐specific products were formed, those formed at lower temperatures were presumably due to
annealing of primers to non‐specific sites on the template. It is unclear why non‐specific products were
formed at temperatures higher than the Ta0PT, but this was a consistent finding” from Rhychlik et. Al
(1990).

L  |‐‐‐‐Primer 1‐‐‐‐‐‐| L  |‐‐‐‐ Primer 2‐‐‐‐‐‐||‐‐‐‐‐Primer 3‐‐‐‐|
1kb    55  56.5   58   59.1   61    62.3   1kb    55   56.5   58    59.1  61   62.3   55   56.5   58    59.1   61    62.3

An abandoned presentation for SURE SHOT 
5/24/2010
CLONING ‐ Atul Kakrana
 A question from last seminar
Can we reamplify a PCR product? Yes, we can

1. Use proofreading enzyme in all the amplifications to avoid mutations and errors especially at primer 
binding sites.
2. Clean amplified DNA  either by PCR purification kit or Gel elution method before using for next round of 
amplification
3. Dilute amplified product before re‐amplification to be used as template.
4. Design primers internal to sequence amplified in first cycle. 

An abandoned presentation for SURE SHOT 
5/24/2010
CLONING ‐ Atul Kakrana
 END OF PRESENTATION

THANK YOU FOR YOUR ALACRITY

AND PRECIOUS TIME

An abandoned presentation for SURE SHOT 
5/24/2010
CLONING ‐ Atul Kakrana