TOPO® XL PCR Cloning Kit, with One Shot® Mach1™ T1 Phage-Resistant Chemically Competent E. coli - FAQs

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 µg/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.

The ends of pCRII and pCR2.1 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 usually 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. If you suspect this is a problem for your clone, here are a few things to try:

1. Increase the annealing temperature of the cycling, or even try a two-stage cycling reaction.
2. Add DMSO to the reaction. This may reduce secondary structure formation. However, beware that it may also affect the fluorescence or some other portion of the reaction.
3. Design primers internal to the insert.
4. Last resort - switch to non-cycling conditions, i.e. manual sequencing using sequenase, S-35-labelled dNTP's. This is perhaps the least convenient, but most effective method.

1. Large inserts should be gel-purified prior to cloning. Gel purification of long PCR products often improves efficiency, as smaller non-specific bands that may not be visible on the gel will interfere with cloning of the insert of interest.

2. Cloning efficiency of gel-purified products can be further increased by avoiding visualization of the bands with UV light. Ethidium bromide and UV light can cause nicking of the gel-isolated DNA, and nicked DNA is a poor substrate for cloning - it is degraded much more efficiently by nucleases in the bacteria.

One option is to purify from a gel stained with SYBR Safe stain and visualized with blue light on the Safe Imager. Data showing improved cloning efficiency after gel purification with this method versus ethidium bromide and UV light is available on the Thermo Fisher Scientific website at www.thermofisher.com/safeimager.<

Another option is to stain the DNA with crystal violet - the pCR-XL TOPO Cloning kits and SNAP UV-Free Gel Purification Kit (Cat. No. K2000-25) use crystal violet instead of ethidium bromide and UV to visualize the PCR product during gel isolation. Crystal violet is also available separately from Sigma (Cat. No. C3886), and can be used for gel purification of PCR products for cloning into any TOPO-adapted vector.

If neither of these options is convenient, try to use a minimal amount of EtBr in the gel, only enough to visualize the fragment of interest. 0.5 µL of a 10 mg/mL stock of EtBr per 100 mL of agarose gel is generally sufficient.

3. Keep the insert:vector molar ratio low, optimally 1:1, a maximum of 3:1 to 5:1 ratio. If the concentration of insert is too high, one end of the PCR product is able to bind to one end of the vector, and the other end of the PCR product would then have to compete with the other free-floating PCR products in the solution for the other end of the vector. This competition becomes more significant as the size of the insert increases.

4. Increase incubation time of the TOPO reaction to either 1 hour, 2 hours, or even overnight incubations at room temperature. These longer incubations have been used without any detrimental effects as long as the Salt solution is used in the TOPO reaction. Longer incubations generally increase the cloning efficiency for longer PCR fragments; shorter fragments show little benefit from these longer incubations.

5. Dilute the reaction to 20 µL, while maintaining the same amount of vector and insert. Increase salt solution volume to 3.7 µL to compensate for the increase in volume, and add sterile distilled water up to 20 µl. Diluting the reaction reduces the competition for the vector ends further.

Any one or combination of the above strategies should improve the efficiencies 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.

It is best practice to remove the enzymes via phenol-chloroform extraction to prevent the proofreading enzyme from removing the A-overhang again after Taq incubation. If not done, TA cloning efficiencies could be 4-10 fold lower. Alternatively, the PCR product can be cleaned up by gel purification or PCR cleanup column.

Phosphorylated products can be TA-cloned, but not TOPO-cloned. This is because the necessary phosphate group is already contained within the Topoisomerase-DNA intermediate complex on the TOPO vectors. The TOPO plasmid has a 3' phosphate, to which Topoisomerase is covalently bound. The PCR product must provide 5'OH and 3'OH groups to react with the Topoisomerase, and presence of an additional phosphate group will inhibit the Topoisomerase-mediated ligation.

The non-TOPO TA Cloning vectors have a 3'OH and a 5' phosphate, and because they involve use of standard ligase, they are compatible with PCR products both with or without 5' and 3' phosphate groups.

Pre-phosphorylated PCR products can be phosphatase-treated (CIP/BIP) to remove the phosphate groups before TOPO cloning - however, cloning efficiency may be decreased compared with an insert that was never phosphorylated at all.

Here is a list of some commonly known PCR enzymes and indication of their compatibility with TA Cloning vectors.

1) Enzymes that leave abundant 3'-A overhangs, directly compatible with TA Cloning:
- Invitrogen Taq Polymerase, Platinum Taq Polymerase, Platinum PCR Super Mix
- Applied Biosystems AmpliTaq, AmpliTaq Gold, and AmpliTaq 360 DNA Polymerase
- Invitrogen SuperTaq and SuperTaq Plus
- Roche Taq Polymerase and Tth Polymerase
- TaKaRa Taq
- Agilent Taq2000 polymerase, Exo-Pfu Polymerase

2) Enzymes that leave partial A overhangs, generally contain mix of Taq and proofreader and may have lower efficiency with TA Cloning:
- Invitrogen Platinum Taq High Fidelity and Platinum PCR Supermix High Fidelity
- Roche Expand System
- TaKaRa LA Taq and Ex Taq

On a typical cloning plate with blue/white screening, there are a few white colonies which do not contain insert. These colonies are usually larger than the other colonies and are due to a deletion of a portion of the plasmid sequence by a rare recombination event (usually from the polylinker to a site in the F1 origin). For the best chance to find a colony with a proper insert, pick clones of a variety of color and pattern for analysis. An insert may generate distinct patterns according to its orientation and size. Even dark blue colonies can have inserts present, if the insert is small and a resulting ORF is in frame with the LacZalpha.

Most of our cloning vectors are offered in a complete format, which includes competent cells. While in most cases other cells can be used with our vectors, we cannot guarantee the results you will get with our cloning vectors if you use your own competent cells. For this reason, most TOPO TA Cloning vectors can only purchased in a complete kit with cells, although there are a few exceptions. Non-TOPO vectors are generally available in multiple formats, with and without cells. To get the most current information on available products, visit the Cloning section of our website under Products & Service.

Zero Background and Zero Blunt vectors are available without competent cells provided, but you should especially careful in choosing competent cells to use with them. These vectors contain the ccdB gene for efficient negative selection of clones without insert, and some E. coli strains are not compatible with the mechanism of negative selection by the lethal activity of the ccdB gene product. In particular, cells with the F' episome have a ccd locus containing the ccdA gene, which prevents ccdB protein cell-killing. Therefore, cells without the F' episome are recommended so that only the CcdB protein will be expressed, and its cell-killing ability will not be inhibited or reduced by CcdA.

The pCRII-TOPO vector is a dual promoter vector. It contains a T7 promoter at the 5' end of the multiple cloning site and an Sp6 promoter at the 3' end. The pCR2.1-TOPO vector only contains the T7 promoter at the 5' end. The dual promoter vector is only of advantage for in vitro transcription studies with the cloned insert. In terms of cloning efficiency, there is no difference between the pCR2.1-TOPO and the pCRII-TOPO plasmids. Both the pCR2.1-TOPO and the pCRII-TOPO vector contain M13 sequencing primer sites to sequence the cloned insert in both directions.

TOP10, TOP10F', DH5a and INValphaF' are some of the strains offered with the Original TA Cloning Kits and the TOPO TA Cloning Kits. E. coli strains DH5a,TOP10, and INValphaF' do not have the lacIq repressor and permit constitutive expression from the lac promoter. With these cells, there is no need to add IPTG (inducer) for blue/white screening with X-gal. In contrast, E. coli strain TOP10F' carries the lacIq repressor and requires IPTG inducer to be added to enable blue/white screening. Please note, the F' episome in the INValphaF' strain is different from other strains in that it does not contain the lacIq repressor. Usually, presence of the F' and lacIq is only an advantage if you work with an insert that is potentially toxic to the host cell. If your insert is (potentially) toxic, we recommend using the TOP10F' cells without adding any IPTG. The lacIq repressor will repress expression from the lac promoter and you won't get blue-white screening, but you will still get colonies. This way you can clone a toxic construct.

TOPO TA cloning kits are also offered with Mach1 T1r competent cells. The Mach1 T1 Phage-Resistant (T1R) E. coli strain is the fastest growing chemically competent strain currently available. Doubling time is approximately 50 minutes, compared with an excess of 74 minutes for other cloning strains. Mach1 colonies are clearly visible within eight hours of plating the transformation mix, enabling you to plate and pick colonies in the same day. With these cells, there is no need to add IPTG (inducer) for blue/white screening.

With a vector containing 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 an insert. X-gal is added to the plate as a substrate for the LacZ-encoded beta-galactosidase enzyme and must always be present to see the blue color in the blue/white screening.

In cases where the lacIq repressor is present (either provided by the host cells on an F' episome, i.e. TOP10F', or expressed from the plasmid), it will repress expression of LacZ from the lac promoter and prevent the formation of the blue color with X-gal. This repression can be reversed by adding IPTG to the media in addition to X-gal, which will inhibit the action of lacIq and re-activate expression from the lac promoter.