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TOPO® Cloning
TABLE OF CONTENTS
PRODUCT DESCRIPTION
SHIPPING CONDITIONS
STORAGE CONDITIONS
STABILITY
QC SPECIFICATIONS
PROTOCOL & APPLICATION NOTES
TOPO vectors
Primer Design considerations
PCR reaction
TOPO Cloning Reaction
Cloning large inserts
Crystal Violet
Selection with the lacz gene
Selection with the ccdB gene
Transformation
Suggestions for increasing the number of primary colonies with Antibiotics
Selection with Antibiotics
Sequencing of TOPO clones
In Vitro Transcription and Translation
Directional TOPO
ALTERNATE PRODUCTS & COMPATIBILITY
PRODUCT DOCUMENTATION
REFERENCES
PRODUCT NAME & CATALOG NUMBER
COMPONENTS
ASSOCIATED PRODUCTS
1
PRODUCT DESCRIPTION
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The TOPO TA PCR cloning kits provide a highly efficient, 5-minute, one-step cloning strategy for the direct insertion of
Taq-amplified PCR products into a plasmid vector. The Zero Blunt TOPO PCR Cloning provides a highly efficient, 5-
minute, one-step cloning strategy for the direct insertion of blunt-end PCR products generated by a proofreading
polymerase into a plasmid vector. Once cloned into any of the TOPO cloning vectors, the clones can be subsequently
sequenced, sub-cloned into other vectors or in-vitro transcribed for labeled probe generation. No ligase, post-PCR
procedures, or PCR primers containing specific sequences are required.
Vaccinia virus Topoisomerase I is a DNA gyrase that binds to duplex DNA at specific sites and cleaves the
phosphodiester backbone after 5�-CCCTT in one strand. The energy from the broken phosphodiester backbone is
conserved by formation of a covalent bond between the 3� phosphate of the cleaved strand and a tyrosyl residue (Tyr-274)
of topoisomerase I. The phospho-tyrosyl bond between the DNA and enzyme can subsequently be attacked by the 5�
hydroxyl of the original cleaved strand, reversing the reaction and releasing topoisomerase.
TOPO cloning takes advantage of the ligating activity of topoisomerase I by covalently binding the enzyme to each end of
a linearized plasmid. The plasmid vectors are supplied linearized with topoisomerase I covalently bound to the 3�-
phosphate of the 3�-Thymidine (at the topoisomerase I-binding sequence YCCTT) via a proprietary process (referred to as
"TOPO-activated" vector). The linearized, TOPO-activated vector is then ready to accept PCR products with compatible
ends as inserts, with topoisomerase facilitating the ligation.
Topoisomerase ligation requires a phosphorylated 3� end and a non-phosphorylated 5� end; i.e. the insert must be non-
phosphorylated at its 5� ends. Once completed with the ligation, the topoisomerase is released from the DNA.
PCR products amplified using standard (non phosphorylated) primers make ideal substrates for the reaction
The vectors pCR4-TOPO, pCR-XL-TOPO, pCR-Blunt II-TOPO, and pCR4Blunt-TOPO allow direct selection of
recombinants via disruption of the lethal E. coli gene, ccdB. These vectors contain the ccdB gene fused to the C-terminus
of the LacZalpha fragment. Ligation of a PCR product disrupts expression of the lacZalpha-ccdB gene fusion permitting
growth of only positive recombinants upon transformation. Cells that contain non-recombinant vector are killed upon
plating. Therefore, blue/white screening is not required.
The pCR4-TOPO and pCR4Blunt-TOPO vectors have been modified to reduce the length of the multiple cloning site in
order to shorten the distance from the primer sites to the insert. This minimizes the amount of vector DNA to be read
before reaching the gene of interest.
The TOPO XL PCR Cloning kit for cloning long PCR products (3-10 kb) makes use of a gel-purification step that
involves visualizing the PCR product on agarose gels containing crystal violet. Thereby the damaging effects of ethidium
bromide in the presence of UV light are avoided, leading to higher cloning efficiencies.
SHIPPING CONDITIONS
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All TOPO PCR Cloning Kits are shipped on dry ice.
STORAGE CONDITIONS
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The box containing the vector should be stored at �20oC, and the box containing the competent E. coli should be stored at
�80oC.
TOPO XL Cloning bag: Store at Room Temp.
The TOPO XL PCR Cloning Kit contains contents with two storage temperatures: Room Temperature and �20oC. Store
Sodium Iodide Solution, Binding Buffer, 4x Final Wash, and TE Buffer at Room temperature. The remainder of this box
is to be stored at -20°C.
STABILITY
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All reagents are guaranteed stable for 6 months when properly stored.
QC SPECIFICATIONS
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2
See Manuals for exact specifications.
PROTOCOL AND APPLICATION NOTES
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TOPO vectors
Primer Design considerations
PCR reaction
TOPO Cloning Reaction
Cloning large inserts
Crystal Violet
Selection with the lacz gene
Selection with the ccdB gene
Transformation
Suggestions for increasing the number of primary colonies with Antibiotics
Selection with Antibiotics
Sequencing of TOPO clones
In Vitro Transcription and Translation
Directional TOPO
TOPO vectors
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Different dyes are added to the TOPO cloning vector solutions during the manufacturing process. The dye is used to
visualize the vector solution during purification of the TOPO-conjugated vector and does not affect the function of the
vector itself. Here is a list of the dyes added to TOPO vectors:
1. TOPO and TOPA TA Vectors (non-directional): Phenol Red. Note: The color should be pink (or yellow) at
room temperature; if it turns blue when PCR product is added then the PCR product buffer is too basic (>9) and
the number of transformed colonies will drop. When the solution is yellow it signifies an acidic pH. At a pH
2.0, TOPO vectors still maintain a high cloning efficiency.
2. Directional TOPO and Zero Blunt TOPO: Bromophenol Blue.
3. The rest of our TOPO vectors are dispensed into aliquots from stored bulks.
All the TOPO vectors have a pUC ori.
The TOPO vectors cannot be electrophoresed on an agarose gel. The vectors contain the Topoisomerase enzyme
conjugated to either end of the vector that inhibits the vector DNA from running into the gel.
The pCRII-TOPO vector contains a T7 promoter at the 5' end of the multiple cloning site and a SP6 promoter at the 3'
end. The pCR2.1-TOPO vector only contains the T7 promoter. The only advantage to having a dual promoter vector is if
one wants to do in vitro transcription studies with the insert. In terms of cloning efficiency, there is no difference between
pCR2.1- TOPO and pCRII-TOPO.
The pLac promoter in all the TOPO vectors is the wild-type pLac that drives expression of the lacZ fragment.
If working with the TOPO vectors that contain the lac promoter and the LacZ alpha fragment (for alpha
complementation), blue/white screening can be used as a tool to select for presence of the insert. X-gal is added to the
plate as a substrate for the LacZ enzyme and must be present for blue/white screening. If the LacIq repressor is present
(either provided by the host cells, for example TOP10F', or expressed from the plasmid) it will repress expression from
the lac promoter thus preventing blue/white screening. Hence in the presence of the LacIq repressor, IPTG must be
provided to inhibit the repressor. Inhibition of LacIq permits expression from the lac promoter for blue/white screening.
There is no dam methylation site (GmATC) overlapping the XbaI site in pCR2.1- and pCRII-TOPO. Therefore, the XbaI
site in these vectors will not be protected from digestion by the restriction endonuclease XbaI regardless of whether the
DNA is isolated from a dam+ or dam- E. coli strain.
pCR4- and pCR4Blunt-TOPO vectors are designed to allow the creation of nested deletions. A comprehensive protocol is
provided in the respective manuals.
Information regarding the antibiotic resistance gene
3
The kanamycin resistance gene in the TOPO vectors are not expressed in mammalian cells. For a gene to be expressed in
a mammalian cell there needs to be a mammalian or suitable viral (SV40, CMV) promoter present. There are no
documented mammalian promoters in the TOPO vectors. The kanamycin resistance gene originated from Tn5 and has the
native promoter.
The kanamycin and ampicillin resistance bicistronic mRNA is transcribed by the native Tn5 promoter that is the strong
kanamycin promoter in pCR2.1- or pCRII-TOPO. The bicistronic kan-amp transcript is terminated at the pUC sequence
downstream of the ampicillin resistance gene.
Primer Design considerations
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Primers can be designed using the OligoPerfectTM Designer software on Invitrogen’s website.
PCR primers should not have 5´-phosphates when cloning into any TOPO vector as the presence of 5�-phosphates inhibit
the TOPO Cloning reaction. Phosphorylated products can be TA-cloned but not TOPO-cloned.
PCR products generated with 5'-biotinylated primers (or any other 5'-label including 5�-Cy5) will not ligate into any of
the TOPO vectors due to steric hindrance
TOPO vectors can be used to clone LUX primer generated PCR products.
PCR reaction
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When doing a TOPO cloning reaction, 2 µl of a PCR reaction containing up to 10% DMSO or 1.3 M betaine will not
interfere with the TOPO reaction. Formamide and high levels of glycerol will inhibit the reaction. These reagents are
usually added to the PCR reaction to enhance the yield of the PCR product, e.g. reduce the effect of secondary structure
or assist in amplification of GC-rich sequences. The effects of tricine or acetamide have not been tested on the TOPO
cloning reaction.
Cloning of Taq DNA polymerase generated PCR products into the Zero Blunt TOPO cloning vectors is not
recommended. Due to the terminal transferase activity of Taq DNA polymerase, PCR products amplified with this
enzyme have 3´-A overhangs. In order to clone these products into the Zero Blunt TOPO vectors and obtain efficient
results, it is first necessary to polish the ends to make them blunt, which usually is not an efficient process.
If using a polymerase mixture containing Taq DNA polymerase and a proofreading polymerase, Taq must be used in
excess of a 10:1 ratio of Taq to the proofreading enzyme to ensure the presence of 3´ A-overhangs on the PCR product. If
the polymerase mixtures do not have enough Taq polymerase or if a proofreading polymerase is used then 3' A-overhangs
can be added following PCR as described in the appendix of the TOPO manuals.
A phenol-chloroform extraction should be done after (re)-generating 3’A-overhangs with Taq polymerase following PCR
using a proofreading polymerase. This is necessary to prevent the proofreading enzyme from removing the A-overhang.
If this is not done, cloning efficiencies are 4-10 fold lower. Alternatively gel purification or a PCR cleanup column can
also clean up the PCR product.
Use fresh PCR products for the TOPO cloning reaction. The cloning efficiency is reduced after as little as 1 day of
storage. Use no more than 2-3 µl of the PCR mixture in the reaction since the high salt content of PCR can inhibit
ligation. The suggested vector: insert molar ratio is 1:1 or 1:3 for most TOPO cloning vectors.
There is some reduction in A-overhangs if the PCR product is gel purified, which along with PCR product loss during the
procedure may slightly reduce total number of colonies. However the percentage of colonies with insert does not change;
it is typically >90% recombinant clones. Gel purification is not required if the gel indicates that the PCR product is clean
with no visible non-specific bands or primer dimmers. It is recommended if the PCR product is >1.5 kb or if non-specific
bands and primer dimers are visible on the gel. Smaller products clone much more efficiently into the vector than larger
products; therefore they should be eliminated from the sample prior to cloning.
TOPO Cloning Reaction
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4
The composition of the 6X Stop solution is 0.3M NaCl, 0.06M MgCl2 and the composition of the 6X Salt solution is
1.2M NaCl, 0.06M MgCl2. Stop solution is only included in the TOPO XL Cloning kit whereas Salt solution is currently
included in all the other TOPO Cloning kits.
The function of the stop/salt solutions is to prevent free topoisomerase from re-binding and nicking the plasmid, which
would reduce the number of colonies from a TOPO reaction. Storage of the TOPO vector plus insert reaction for 1 week
at 4oC has shown no detectable decrease in the cloning efficiency of the TOPO reaction as >95% of the colonies have
insert, however the total number of colonies was decreased by 10-fold. Storage of the TOPO reaction mix overnight at
4oC showed little to no decrease in the number of colonies when compared to fresh TOPO reaction mix.
When cloning into either pCR2.1- or pCRII-TOPO, if an ampicillin-resistant non-linearized plasmid is used as a template
for PCR, and gel purification of the PCR product is not done there is a high possibility of propagating the parental
template when selecting with ampicillin. The supercoiled template plasmid will transform the competent cells much more
efficiently than the TOPO-cloned plasmid. Options to prevent this include using kanamycin as the selective antibiotic
since both pCRI2.1 and pCRII-TOPO have ampicillin and kanamycin resistance genes or gel purify the insert before
cloning.
Cloning large inserts (5-10 kb)
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Regular TOPO TA Cloning kits are efficient for PCR products up to an estimated 4 kb. For cloning large inserts (3-10 kb) we
recommend the TOPO XL kit. For cloning of large inserts in the TOPO TA vectors, below are a few recommendations:
1. The pCR-XL TOPO cloning kit uses crystal violet instead of Ethidium Bromide (EtBr) to visualize the PCR for gel
isolation. This reduces the nicking of gel-isolated fragments often associated with EtBr-stained gels. Nicked DNA is a
poor substrate for cloning and is degraded much more efficiently by nucleases in bacteria. If crystal violet is not
available, use a very low amount of EtBr in the gel, only enough to visualize the fragment of interest for purification. A
half a microliter of a 10mg/mL stock of EtBr per 100mL of agarose gel is sufficient.
2. Increase incubation time of the TOPO reaction to at least 30 minutes or up to 1 hour, 2 hours, or overnight incubations at
room temperature. The longer incubations have been used without any detrimental effects as long as the Salt solution is
used in the TOPO reaction. Longer incubations can increase the efficiency for longer PCR fragments; shorter fragments
show little benefit from these longer incubations.
3. Keep the insert:vector molar ratio low, optimally 1:1 or even a 3:1 to 5:1. If the concentration of insert is too high, one
end of the PCR product is capable of binding to one end of the vector, and the other end of the PCR product may compete
with the other free-floating PCR products in solution for the other end of the vector. This competition becomes more
significant as the size of the insert increases and can reduce the number of colonies observed following transformation.
4. Dilute the reaction to 20 µl, while maintaining the same amount of vector and insert. Increase the volume of the salt
solution to 3.7 µl to compensate for the increase in volume. Diluting the reaction reduces the competition for the vector
ends further.
Any single or combination of the above strategies should improve the efficiency of cloning larger inserts in a TOPO reaction.
Keep in mind that the TOPO reaction does have a strong size bias, and that any smaller fragments present in the PCR reaction
would clone much more efficiently than the fragment of interest, even if the smaller fragment is not readily visible on a gel.
Therefore, it is highly recommended that the PCR product of interest be gel-purified.
The inclusion of salt solution in the TOPO Cloning reaction increases the number of transformants 2- to 3-fold. It has been also
observed that in the presence of salt, incubation times of greater than 5 minutes will also increase the number of transformants.
Salt prevents topoisomerase I from rebinding and potentially nicking the DNA after ligating the PCR product and dissociating
from the DNA.
Crystal Violet Stain
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Crystal Violet and 6X Crystal Violet Loading Buffer are found in the TOPO XL PCR Cloning Kits.
In side-by-side cloning experiments, long PCR products that were purified after visualization with crystal violet yielded
94% recombinant colonies versus only 60% with ethidium bromide staining an UV light visualization.
5
To increase the intensity of the crystal violet-stained gel, allow the gel to lie for a couple of hours in crystal violet stain
(45 µl of 2 mg/ml stain in 100 ml sterile water or 1-10 ug/ml stain in 0.1X TAE) and it will stain a little darker. Place the
gel over a white background when excising the band to improve visibility.
Selection with the ccdB genes
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TOPO vectors containing the LacZ-ccdB cassette allows direct selection of recombinants via disruption of the lethal E.
coli gene, ccdB. Ligation of a PCR product disrupts expression of the lacZα-ccdB gene fusion permitting growth of only
positive recombinants upon transformation. Cells that contain non-recombinant vector are killed upon plating. Therefore,
blue/white screening is not required.
A minimum insertion of 150 bp is needed in order to ensure disruption of the ccdB gene and prevent cell death.
(Reference: Bernard et al.,1994. Positive-selection vectors using the F plasmid ccdB killer gene. Gene 148: 71-74.)
Strains that contain an F plasmid, such as TOP10F�, are not recommended for transformation and selection of
recombinant clones in any TOPO vector containing the ccdB gene. The F plasmid encodes the CcdA protein, which acts
as an inhibitor of the CcdB gyrase-toxin protein. The ccdB gene is also found in the ccd (control of cell death) locus on
the F plasmid. This locus contains two genes, ccdA and ccdB, which encode proteins of 72 and 101 amino acids
respectively. The ccd locus participates in stable maintenance of F plasmid by post-segregational killing of cells that do
not contain the F plasmid. The CcdB protein is a potent cell-killing protein when the CcdA protein does not inhibit its
action. The half-life of the CcdA protein is shorter than that of the CcdB protein. Persistence of the CcdB protein leads to
death of bacterial segregants that do not contain the F plasmid. Overexpression of the CcdB protein causes cell death
when sufficient CcdA does not prevent its action. In theory, and since the half-life of the CcdA protein is much shorter
than that of CcdB, there should be no difference in the growth or in the selection between E. coli strains that carry an F
plasmid and those that do not.
Selection with the lacz gene
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If working with a vector that contains the lac promoter and the LacZ alpha fragment (for alpha complementation),
blue/white screening can be used as a tool to select for presence of the insert. X-gal is added to the plate as a substrate for
the LacZ enzyme and must always be present for blue/white screening.
The minimum insert size needed to completely disrupt the lacZ gene is >400bp
If the lacIq repressor is present (either provided by the host cells, for example TOP10F', or expressed from the plasmid) it
will repress expression from the lac promoter thus preventing blue/white screening. Hence in the presence of the lacIq
repressor, IPTG must be provided to inhibit the lacIq. Inhibition of lacIq permits expression from the lac promoter for
blue/white screening.
X-gal (also known as 5-bromo-4-chloro-3-indolyl beta-D-glucopyranoside) is soluble in DMSO or DMF, and can be
stored in solution in the freezer for up to 6 months. Protect the solution from light.
Final concentration of X-gal and IPTG in agar plates: Prior to pouring plates, add X-gal to 20 µg/ml and IPTG to 0.1 mM
to the medium. When adding directly on the surface of the plate, add 40µl X-gal (20 mg/ml stock) and 4 µl IPTG (200
mg/ml stock).
Transformation
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TOPO TA Kits are sold in combination with DH5alpha, Mach1-T1R, TOP10 or TOP10F' competent E.coli. E. coli strains
DH5alpha, and TOP10 do not have the lacIq repressor. Therefore, these strains permit constitutive expression from the
lac promoter and there is no need to add IPTG (inducer) for blue/white screening. E. coli strain TOP10F' carries the lacIq
repressor and IPTG is required for expression from the lac promoter for blue/white screening. If the insert is potentially
toxic to the host cell, TOP10F' cells are recommended without no addition of IPTG. The lacIq repressor will repress
expression from the lac promoter, so blue-white screening cannot be used. If low numbers of transformants or clones
with deleted inserts are observed, this may be caused by the instability of the insert DNA in TOP10 E. coli. In this case,
E. coli strains such as Stbl2, Stbl3, or Stbl4 have been shown to support the propagation of DNA with multiple repeats,
6
retroviral sequences, and DNA with high GC content better than other strains (Trinh, et al., (1994) FOCUS, 16-3, p78-
80).
If no growth is observed from picked colonies in mini-prep liquid culture, one possible explanation could be toxicity
associated with the insert. This toxicity does not affect slow growing cells on solid medium but is much stronger in faster
growth conditions like liquid medium. Suggestions:
1. Use TOP10F� or any other strain with the LacIq repressor
2. Try using any other strain appropriate for cloning.
3. Lower growth temperature to 27-30oC and grow the culture longer
4. Another possibility to explain lack of growth is possible phage contamination. In this situation we recommend
using an E. coli strain that is T1 phage resistant like DH5alpha-T1R.
The biosafety level of the pcR2.1-TOP10 host-vector system is EK1 for Biological Containment. EK1 refers to Standard
laboratory E. coli K12 host strain and compatible plasmid and virus vectors.
Both R408 and M13K07 have been used as helper phage in-house with similar results for single strand DNA rescue.
M13K07 is often preferred since it can be selected with kanamycin.
Suggestions for increasing the number of primary colonies
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Longer incubation of the TOPO cloning reaction at room temperature (5 fold more colonies are observed from 4 hrs to
overnight incubation) provided that the 6X Salt solution is added to the reaction.
Electroporation can also give significant increases in colony numbers; often 10-20 fold higher. However, if doing
electroporation, it is important that the TOPO-reaction mix contains diluted Salt solution or for best results precipitated
prior to transformation. For high primary transformants by electroporation it is recommended to:
1) Perform the TOPO reaction for at least 3 hours at 37oC,
2) Add 100 µl double diH2O to the 6 µl TOPO reaction and incubate 10 more minutes at 37oC.
3) Precipitate by adding 10 µl 3 M Na-Acetate, 2 µl 20 µg/µl glycogen, 300 µl 100% ethanol. Place on
dry ice or �80oC for 20 min, spin at top speed in a microcentrifuge at 4oC for 15 min. Wash pellet with
800 µl 80% ethanol, spin at top speed for 10 min, pour off ethanol, spin 1 min and remove remaining
ethanol without disturbing pellet. Dry pellet (air-dry or speed-vac).
4) Resuspend pellet in 10 µl ddH2O and electroporate 3.3 µl of resuspended DNA according to a normal
electroporation protocol.
This electroporation protocol can yield up to 20 fold more colonies than chemical transformation of an equivalent TOPO-
reaction. The addition of the 100 µl ddH2O followed by 10 min incubation is not absolutely necessary, but it sufficiently
dilutes the reaction and may help inactivate topoisomerase so that it is more easily electroporated.
Selection with Antibiotics
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When using Zeocin as the selection antibiotic do not transform an E. coli strain that has Tn5 in its genotype since Tn5
confers resistance to zeocin.
If small satellite colonies are observed on LB+ Ampicillin plates it is most likely caused due to degradation of the
ampicillin. If the LB-Amp plates are > 2 weeks old, there is a high possibility of the Ampicillin being degraded even
when stored at +4oC. The small white colonies are just untransformed cells that grow on LB with degraded Ampicillin.
In order to circumvent such a scenario, here are 3 suggestions;
1. Plate cells at a lower density
2. Use fresh LB-Amp plates or replace Ampicillin with Carbenicillin (cat# 10177012). Carbenicillin is prepared
and handled in the same way as Ampicillin and the final concentration used is typically 100 ug/ml. Plates
containing both antibiotics (50 ug/ml Ampicillin and 50 ug/ml Carbenicillin) can also be used.
3. The plates should not be incubated for more than 20 hours at 37oC. Beta-lactamase, the enzyme produced from
the Ampicillin-resistance gene, is secreted from the Amp-resistant transformants and inactivates the antibiotic in
the area surrounding the transformant colony. This inactivation of the selection agent allows satellite colonies
(which are not truly Amp-resistant) to grow. This is also true if Carbenicillin is being used.
7
Sequencing of TOPO clones
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The ends of the TOPO vectors are palindromic sequences since both ends must accommodate the TOPO binding sites.
Therefore there is an inherent potential for secondary structure once the vector is ligated. This does not usually affect
sequencing or PCR results, as cycling conditions are sufficient to overcome potential secondary structures that might
form. However, certain inserts might enhance the formation of such secondary structures by the regions flanking the
cloning site. Strong secondary structures may inhibit the cycle sequencing reactions commonly used in automated
sequencing. There are a few things to try:
1. Increase the annealing temperature of the cycling; even try a two-stage cycling reaction.
2. Add DMSO to the reaction. This may reduce secondary structure formation. However it may also affect the
fluorescence or some other portion of the reaction.
3. Design primers internal to the insert.
4. As a last resort, switch to non-cycling conditions; i.e. manual sequencing using Sequenase and 35S-labelled
dNTP's. This is perhaps the least convenient, but most effective method.
Factors to consider that can influence the quality of the sequencing reaction include clone contamination (more than one
clone mixed together), quality (protein, salt, or EDTA contamination), and quantity of DNA. Another important factor is
the base composition of the insert; high A/T, G/C, homobase runs or stem/loop structures can all potentially affect the
sequencing reaction. A suggestion that sometimes helps is to PCR the insert and purify it with a PCR purification kit.
The linear structure of a PCR product helps with denaturation and sequencing read-through.
In Vitro Transcription and Translation
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pCRII-TOPO and pCR-Blunt II-TOPO contain both T7 and SP6 promoters whereas pCR2.1-TOPO, and pCR-XL-TOPO
only have the T7 promoter on the reverse strand of the vector. pCR4- and pCR4Blunt-TOPO contain the T3 and T7
promoters.
The T7 and SP6 promoters are fully functional promoters and can be used in in vitro transcription for generation of
labeled probes or other purposes requiring RNA transcripts based on the insert.
The TOPO Cloning vectors are not recommended for in vitro translation. There are one or more start codons (ATG) on
either side of the cloning sites, which may interfere with attempts to translate an open reading frame in the insert. The
two major factors influencing translation in vitro from one of the undesirable upstream ATGs are: (1) the Kozak
sequence around the ATG (mainly an "A" at position -3 where the "A" in the ATG is +1) and (2) a cap on the 5' end of
the message. The Kozak sequence can be determined simply by looking at the printed sequence of the vector. In the
pCRII-TOPO sequence there is one ATG in a message made from the Sp6 promoter before the PCR insert site and there
are 3 from T7 that are in two of the three reading frames. As mentioned above, none of these four ATGs have an "A" at
the �3 position. Simply adding or removing the correct number of bases from the PCR primers to adjust the frame can
avoid the problem of translating the wrong frame. In vitro transcripts can be capped by doing the synthesis in the
presence of artificial caps, which are commercially available.
Directional TOPO
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For cloning into Directional TOPO vectors, the gene of interest has to be amplified with a proofreading polymerase such
as AccuPrime Pfx or Platinum Pfx that leaves blunt ends. PCR mixes that contain Taq polymerase are not recommended
for D-TOPO cloning.
AccuPrime Pfx or AccuPrime Pfx Supermix is recommended for D-TOPO cloning.
When performing directional TOPO Cloning, the molar ratio of PCR product: TOPO vector used in the reaction is critical
to its success. To obtain the highest TOPO Cloning efficiency, use a 0.5:1 to 2:1 molar ratio of PCR product: TOPO
vector. The TOPO Cloning efficiency decreases significantly if the ratio of PCR product: TOPO vector is <0.1:1 or >5:1.
These results are generally obtained if too little PCR product is used (i.e. PCR product is too dilute) or if too much PCR
product is used in the TOPO Cloning reaction. If the yield of the PCR product has been quantitated, the concentration of
the PCR product may need to be adjusted before proceeding to TOPO Cloning. For pENTRâ„? TOPO vectors, using 1-5 ng of a
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1 kb PCR product or 5-10 ng of a 2 kb PCR product in a TOPO Cloning reaction generally results in a suitable number of colonies.
PRODUCT DOCUMENTATION
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Brochures Citations Cell lines
COA FAQ Licensing
Manuals MSDS Newsletters
Vector Data
REFERENCES
(back to Table of Contents)
Expressions 6.6, p. 14, November 1999
Bernard, P., Gabant, P., Bahassi, E. M., and Couturier, M. (1994). Positive Selection Vectors Using the F Plasmid ccdB
Killer Gene. Gene 148, 71-74.
Shuman, S. (1991). Recombination Mediated by Vaccinia Virus DNA Topoisomerase I in Escherichia coli is Sequence
Specific. Proc. Natl. Acad. Sci. USA 88, 10104-10108.
Shuman, S. (1994). Novel Approach to Molecular Cloning and Polynucleotide Synthesis Using Vaccinia DNA
Topoisomerase. J. Biol. Chem. 269, 32678-32684
PRODUCT NAME AND CATALOG NUMBERS
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Vector Competent
Vector Catalog Reaction Competent E.coli Cells Box part cells part
number Size number number
pCR2.1-TOPO K450001 20 One Shot Chemically competent TOP10 450641 440301
(TOPO TA
Cloning Kit)
K450040 40 One Shot Chemically competent TOP10 450641 440301
One Shot Chemically competent DH5α-T1R
K452001 20 450641 440097
One Shot Chemically competent DH5α-T1R
K452040 40 450641 440097
K455001 20 One Shot Chemically Competent TOP10F� 450641 440300
K455040 40 One Shot Chemically Competent TOP10F� 450641 440300
One Shot Chemically Competent Mach1-T1R
K451020 20 450641 C862003
K456001 20 One Shot Electrocompetent TOP10 450641 440035
K456040 40 One Shot Electrocompetent TOP10 450641 440035
K4500480 480 Chemically Competent TOP10 –Multishot* 450084 440038
K450005 480 Chemically Competent TOP10 –Multishot 450084 440091
Strip Well**
pCRII-TOPO K460001 20 One Shot Chemically Competent TOP10 450640 440301
(TOPO TA
Cloning Kit Dual
Promoter)
K460040 40 One Shot Chemically Competent TOP10 450640 440301
One Shot Chemically Competent DH5α-T1R
K462001 20 450640 440097
One Shot Chemically Competent DH5α-T1R
K462040 40 450640 440097
K465001 20 One Shot Chemically Competent TOP10F� 450640 440300
K465040 40 One Shot Chemically Competent TOP10F� 450640 440300
One Shot Chemically Competent Mach1-T1R
K461020 20 450640 C862003
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K466001 20 One Shot Electrocompetent TOP10 450640 440035
K466040 40 One Shot Electrocompetent TOP10 450640 440035
K4600480 480 Chemically Competent TOP10 –Multishot* 450083 440038
K460005 480 Chemically Competent TOP10 –Multishot 450083 440091
Strip Well**
pCR4-TOPO K4575J10 10 One Shot Chemically Competent TOP10 440012 450071
(TOPO TA
Cloning Kit for
Sequencing)
K457501 20 One Shot Chemically Competent TOP10 450030 440301
K457540 40 One Shot Chemically Competent TOP10 450030 440301
One Shot Chemically Competent DH5α-T1R
K459501 20 450030 440097
One Shot Chemically Competent DH5α-T1R
K459540 40 450030 440097
Chemically Competent Mach1-T1R
K453020 20 450030 C862003
K458001 20 One Shot Electrocompetent TOP10 450030 440035
K458040 40 One Shot Electrocompetent TOP10 450030 440035
K4575480 480 Chemically Competent TOP10 –Multishot* 450090 440038
K457505 480 Chemically Competent TOP10 –Multishot 450090 440091
Strip Well**
pCR-XL-TOPO K470010 10 One Shot Electrocompetent TOP10 450008 440033
(TOPO XL PCR
Cloning Kit)
K470020 20 One Shot Electrocompetent TOP10 450008 440033
K475010 10 One Shot Chemically Competent TOP10 450008 440012
K475020 20 One Shot Chemically Competent TOP10 450008 440012
One Shot Chemically Competent Mach1-T1R
K703020 20 450008 C862003
One Shot OmniMAX 2-T1R
K803020 20 450008 C854003
pCR8/GW/ K250020 20 One Shot Chemically Competent. Top10 450642 440301
TOPO
One Shot Chemically Competent Mach1-T1R
K252020 20 450642 C862003
Vector Competent
Vector Catalog Reaction Competent E.coli Cells Box part cells part
no. Size number number
pCRBluntII- K280020 20 One Shot Chemically Competent TOP10 450245 440301
TOPO (Zero
Blunt TOPO PCR
Cloning Kit)
K280040 40 One Shot Chemically Competent TOP10 450245 440301
One Shot Chemically Competent DH5α-T1R
K282020 20 450245 440097
One Shot Chemically Competent DH5α-T1R
K282040 40 450245 440097
One Shot Chemically Competent Mach1-T1R
K283020 20 450245 C862003
K286020 20 One Shot Electrocompetent TOP10 450245 440035
K286040 40 One Shot Electrocompetent TOP10 450245 440035
pCR4Blunt- K2875J10 10 One Shot Chemically Competent TOP10 450159 440012
TOPO (Zero
Blunt TOPO PCR
Cloning Kit for
Sequencing)
K287520 20 One Shot Chemically Competent TOP10 450031 440301
K287540 40 One Shot Chemically Competent TOP10 450031 440301
K2875480 480 Chemically Competent TOP10 –Multishot* 450089 440038
One Shot Chemically Competent DH5α-T1R
K289520 20 450031 440097
One Shot Chemically Competent DH5α-T1R
K289540 40 450031 440097
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One Shot Chemically Competent Mach1-T1R
K283520 20 450031 C862003
K288020 20 One Shot Electrocompetent TOP10 450031 440035
K288040 40 One Shot Electrocompetent TOP10 450031 440035
* Multishot: Cells are provided pre-aliquoted (15 µl/well) in 96-well plates (MultiShot format) to allow addition of the TOPO
Cloning reaction to the cells. Includes 5 x 96 well plates and 1 Test plate with 12 wells of cells (1 row).
COMPONENTS
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All kits have the following: Manual, Vector Box, and Competent cells.
Vector Box: The table below describes the components in the TOPO Vector and Cloning Reagents Box.
Note: 1 box for 20-rxn or 10-rxn kit, and 2 boxes for 40-rxn kit.
Our TOPO packaging boxes are from a company in the US called Royal Paper Box. Our contact is Darryl Carlson (323) 728-
7041.
Amount in
Item (part number) Concentration Amount HTP Kits
20 µl (20 reaction kit)
VECTOR : 10 ng/µl plasmid DNA in: 500 µl
pCR2.1-TOPO (460801) 50% glycerol, or
HTP-pCR2.1-TOPO (460267) 50 mM Tris-HCl, pH 7.4 (at 15 µl (10 reaction kit)
pCRII-TOPO (460800) 25°C),
HTP-pCRII-TOPO (460269) 1 mM EDTA,
pCR4-TOPO (460204) 10 rxn 2 mM DTT,
pCR4-TOPO (460080) 20 rxn 0.1% Triton X-100,
HTP-pCR4-TOPO (460270) 100 ug/ml BSA,
pCR-Blunt II-TOPO phenol red or bromophenol blue
(460096)
pCR4Blunt-TOPO ( 450031)
10X PCR Buffer (460121) 100 mM Tris-HCl, pH 8.3 (at 100 µl --
Note: Not Included with TOPO 42°C)
Blunt vectors 500 mM KCl
25 mM MgCl2
0.01% gelatin
dNTP Mix (460122) 12.5 mM dATP, 12.5 mM dCTP, 10 µl 300 µl
12.5 mM dGTP, 12.5 mM dTTP,
neutralized at pH 8.0 in water
6X Salt Solution (460205) 1.2 M NaCl, 0.06 M MgCl2 50 µl 500 µl
M13 Reverse Primer (460690) 0.1 ug/µl in TE Buffer 20 µl 20 µl
(385 pmoles supplied)
M13 Forward (-20) Primer, (460691) 0.1 ug/µl in TE Buffer 20 µl 20 µl
(407 pmoles supplied)
T3 primer (NOTE: Only with pCR4 0.1 ug/µl in TE Buffer, pH 8 20 µl 20 µl
Vector) (460029) (329 pmoles supplied)
T7 primer (NOTE: Only with pCR4 0.1 ug/µl in TE Buffer, pH 8 20 µl 20 µl
Vector) (460076) (328 pmoles supplied)
Control Template (460118) 0.1 ug/µl in TE Buffer 10 µl --
Control PCR Primers (460100) 0.1 ug/µl each in TE Buffer, pH 8 10 µl --
Sterile Water (460113) -- 1 ml 3 x 1 ml
One Shot Reagents: The table below describes the items included in the One Shot® competent cell kit. Store at -80°C.
Note: 1 box for 20-rxn or 10-rxn kit, and 2 boxes for 40-rxn kit.
Item (part number) Composition Amount in Amount in
20 rxn kit HTP kit
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SOC Medium (460700) (may be 2% Tryptone, 0.5% Yeast Extract, 6 ml 5 x 15 ml
stored at +4°C or room temperature) 10 mM NaCl, 2.5 mM KCl, 10
mM MgCl2, 10 mM MgSO4,
20 mM glucose
DH5α-T1R, TOP10, TOP10F�, Chemically Competent 21 x 50 µl *TOP10 in
Mach1-T1R Chemically Competent 11 x 50 µl Multishot
Chemically Competent or formats (see
Electrocompetent below)
10 µl 10 µl
pUC18 (460124) 10 ng/µl in TE, pH 8
OR
pUC19 (460018) Control 50 µl
10 pg/µl in TE, pH 8 50 µl
HTP MultiShot: 5 x 96 well plates (15ul per well) test plate with 12 wells of cells (1 row)
* Multishot: Cells are provided pre-aliquoted (15 µl/well) in 96-well plates (MultiShot format) to allow addition of the TOPO
Cloning reaction to the cells. Includes 5 x 96 well plates and 1 Test plate with 12 wells of cells (1 row).
** Multishot Strip well: Cells are provided pre-aliquoted (50 µl/well) in 96-well plates (MultiShot format) to allow addition of the
TOPO Cloning reaction to the cells. Includes 5 x 96 well plates.
Sequence of Primers:
Primer Sequence pMoles Supplied
M13 Reverse 5´-CAGGAAACAGCTATGAC-3´ 385
M13 Forward (-20) 5´-GTAAAACGACGGCCAG-3´ 407
T3 5´-ATTAACCCTCACTAAAGGGA-3´ 329
T7 5´-TAATACGACTCACTATAGGG-3´ 328
TOPO XL PCR Cloning Kit (part number 450008)
Item (part number) Concentration Amount
pCR-XL-TOPO (460088) 10 ng/µl plasmid DNA in: 1 tube of 11 µl
50% glycerol; 50 mM Tris-HCl, pH 7.4 (at 25°C);
1 mM EDTA; 2 mM DTT; 0.1% Triton X-100;
100 µg/ml BSA; phenol red
50 mM dNTPs (460122) 12.5 mM dATP; 12.5 mM dCTP; 12.5 mM dGTP; 10 µl
12.5 mM dTTP; neutralized at pH 8.0 in water
6X TOPO Cloning Stop 0.3 M NaCl; 0.06 M MgCl2 25 µl
Solution (460066)
XL Control PCR Template 25 ng/µl in TE Buffer 10 µl
(460091)
XL Control PCR Primers 0.2 µg/µl in TE Buffer (0.1 µg/ml each) 10 µl
(460078)
M13 Forward (-20) Primer 0.1 µg/µl in TE Buffer 20 µl
(460691)
M13 Reverse Primer (460690) 0.1 µg/µl in TE Buffer 20 µl
Sodium Iodide Solution 6.6 M Sodium iodide; 16 mM Sodium sulfite 5 ml
(460090)
Binding Buffer (460087) 7 M Guanidinium HCl 7 ml
4X Final Wash (460085) 400 mM NaCl 2.5 ml
TE Buffer, pH 8 (460116) 10 mM Tris-HCl; 1 mM EDTA, pH 8 2 ml
TOPO XL Cloning bag (part number 450006)
Item Concentration Amount
S.N.A.P. Purification -- 11
Columns 850036
S.N.A.P. Collection Vials -- 11
220026
Crystal Violet 460089 2 mg/ml 0.5 ml
12
6X Crystal Violet Loading 30% Glycerol; 20 mM EDTA; 100 µg/ml Crystal 250 µl
Buffer 460077 Violet
ASSOCIATED PRODUCTS
(back to Table of Contents)
Product Amount Catalog number.
One Shot TOP1- Electrocompetent E. coli 10 reactions C404050
20 reactions C404052
10 reactions C404003
One Shot TOP10 Chemically Competent E. coli 20 reactions C404003
40 reactions C404006
One Shot MAX Efficiency DH5α-T1R Chemically Competent 20 reactions 12297016
E. coli
One Shot Mach1-T1R Chemically competent E. coli 20 reactions C862003
One Shot TOP10F� Chemically competent E. coli 20 reactions C303003
40 reactions C303006
S.N.A.P. MidiPrep Kit 20 reactions K191001
PureLink HQ Mini Plasmid Purification Kit 100 reactions K210001
PureLink PCR Purification Kit 50 reactions K310001
Ampicillin 200 mg 11593019
5 gm 11815024
25 gm 11815032
Kanamycin Sulfate 100 ml (10 mg/ml) 15160054
Carbenicillin 5 gm 10177012
Zeocin 1 gm R25001
5 gm R25005
X-gal 100 mg 15520034
1 gm 15520018
IPTG 1 gm 15529019
S.O.C. Medium 10 x 10 ml 15544034
Platinum Taq DNA Polymerase 100 reactions 10966018
500 reactions 10966034
Platinum Taq DNA Polymerase High Fidelity 100 reactions 11304011
500 reactions 11304029
AccuPrime Pfx DNA Polymerase 200 rxns 12344024
1000 rxns 12344032
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