Long Fragment Cloning | Thermo Fisher Scientific

TOPO XL-2 Complete PCR Cloning Kit

Confidently conquer cloning of long DNA fragments

Long-fragment cloning can be challenging for many reasons including the limitations of traditional molecular biology reagents. The Invitrogen™ TOPO™ XL-2 Complete PCR Cloning Kit provides all the necessary elements to overcome these limitations and enable precise, efficient, and simple cloning of long PCR products up to 13 kb. You can achieve higher cloning efficiencies in just 5 minutes at your benchtop when utilizing the new linearized and topoisomerase I–activated pCR™-XL-2-TOPO™ vector, which is compatible with high-fidelity blunt-end PCR fragments produced by the Invitrogen™ Platinum™ SuperFi™ Green PCR Master Mix.

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Kit contents

The Complete PCR Cloning Kit includes:

TOPO XL-2 PCR Cloning Kit—contains the pCR™-XL-2-TOPO vector
Platinum SuperFi Green PCR Master Mix—features a proofreading DNA polymerase (with >100x the fidelity of Taq polymerase) with high processivity to generate accurate, long amplicons, and an inert density ingredient that allows you to load directly onto an agarose gel for gel resolution and extraction
PureLink™ Quick Gel Extraction and PCR Purification Combo Kit—designed to purify DNA fragments in less than 30 minutes from agarose gels or direct PCR purification using a silica-based spin cartridge
One Shot™ OmniMAX™ 2 T1^R Chemically Competent E. coli Cells—an improved high-efficiency chemically competent cell line, perfect for use in all cloning applications

Figure 1. The pCR-XL-2-TOPO vecto r.

The pCR-XL-2-TOPO vector (Figure 1) includes:

ccdB gene for positive selection
EcoRI site flanking the PCR product insertion site for easy excision of inserts
Ampicillin and kanamycin resistance genes for your choice of antibiotic selection
T7 promoter/priming site for in vitro transcription
T7, T3, and M13 forward and reverse primer sites for sequencing

Advantages of the TOPO XL-2 Complete PCR Cloning Kit

Improved cloning efficiency, especially for long fragments

The TOPO XL-2 Complete PCR Cloning Kit is optimized to provide the highest cloning efficiencies for extra-long PCR fragments, as illustrated in Figures 2, 3, and 4.

Figure 2. The TOPO XL-2 Complete PCR Cloning Kit has superior cloning efficiency for a broad range of different sizes of targets. Lambda genomic DNA PCR fragments ranging from 1 kb to 13 kb were cloned using the TOPO XL-2 cloning kit.

Figure 3. The TOPO XL-2 cloning kit shows superior cloning efficiency compared to the TOPO XL kit. Recommended protocols for each kit were followed for cloning a 7 kb control insert.

Figure 4. The TOPO XL-2 cloning kit has superior cloning efficiency compared to an In-Fusion® Cloning kit (Clontech) over a broad range of target sizes. Human and lambda genomic DNA targets were cloned following the protocols given in the respective kits’ manuals. Cloning efficiency was determined by colony PCR using an internal primer that spans either a 3 kb (human) or ~5 kb (lambda) region of the cloned targets. The high-fidelity polymerase enzyme from the In-Fusion Cloning kit was unable to amplify the human 10 kb DNA target. (See Figure 7 in “Improved sequence accuracy and specificity of inserts”.)

Improved sequence accuracy and specificity of inserts

Platinum SuperFi DNA Polymerase is a proofreading DNA polymerase that combines superior fidelity with the trusted Invitrogen™ Platinum™ hot-start technology, designed for the highest success in PCR. Featuring >100x the fidelity of Taq polymerase, Platinum SuperFi Green PCR Master Mix, included in the TOPO XL-2 PCR Cloning Kit, is ideally suited for cloning, mutagenesis, and other applications benefiting from superior sequence accuracy, such as working with long DNA fragments.

Figure 5. Platinum SuperFi DNA Polymerase exhibits superior fidelity. Relative fidelity values of different DNA polymerases. Polymerase fidelity was measured by next-generation sequencing. The background level of experimental error was estimated from PCR-free library sequencing data. The polymerase fidelities were normalized to that of Taq polymerase.

Figure 6. Platinum SuperFi DNA Polymerase has higher specificity in amplifying larger targets than the Invitrogen™ Elongase™ Enzyme Mix. Targets of 1 kb, 3 kb, 7 kb, 10 kb, and 13 kb in length from lambda genomic DNA were amplified following the manufacturers’ standards for PCR cycling parameters. The Elongase Enzyme Mix is recommended for the TOPO XL PCR Cloning Kit, and Platinum SuperFi DNA Polymerase is provided with the TOPO XL-2 Complete PCR Cloning Kit.

Figure 7. Platinum SuperFi DNA Polymerase shows greater specificity for both human and lambda gDNA targets than Clontech’s high-fidelity (HiFi) polymerase. Amplification protocols were followed per manufacturers’ recommendations. From a 50 µL PCR reaction, 6.5 µL of the human gDNA targets and 4 µL of the lambda gDNA targets were loaded into a 1% agarose gel stained with EtBr.

Improved DNA yield

The PureLink™ Quick Gel Extraction and PCR Purification Combo Kit offers the ability to quickly perform both a gel extraction and a PCR purification with a single kit. The isolated DNA is then ready to use for cloning. By including the PureLink kit in the TOPO XL-2 Complete PCR Cloning Kit, the total yield of DNA is greatly improved due to less handling, a major advantage when working with larger DNA fragments (Figure 8).

Figure 8. The PureLink Quick Gel Extraction and PCR Purification Combo Kit shows greater total DNA yields than the Invitrogen™ S.N.A.P.™ Gel Purification Kit, even for larger fragment sizes. Gel-purified lambda PCR products of various sizes were amplified with Platinum SuperFi 2X Green Master Mix. The same starting material was used for equivalent gel purification using PureLink (provided with TOPO XL-2 kits) and S.N.A.P. (provided with TOPO XL kits) kits, for comparison. Purification protocols were followed as stated in the manuals. Total DNA yield amounts were quantified and compared. Percentages indicate the increase in yield obtained from the PureLink kit compared to the S.N.A.P. kit.

Competent cells with improved transformation efficiency

OmniMAX™ 2 T1^R competent cells offer the highest transformation efficiency (> 5 x 10⁹ transformants/µg pUC19) of any chemically competent E. coli cells in the Invitrogen™ One Shot™ format (Figure 9). These highly versatile cells also provide efficient transformation of highly methylated DNA, since OmniMAX 2 T1^R cells lack the E. coli K12 restriction systems (mcrA Δ(mrr hsdRMS mcrBC)). In addition, the strain has the tonA genotype, which confers resistance to T1 and T5 phage infection. This protects your samples and minimizes the possibility of downtime in your lab due to phage contamination.

Figure 9. The chemically competent OmniMAX™ 2 T1^R cells yield higher numbers of colony forming units (cfu) than TOP10 cells, following transformation of cloned DNA fragments. Clones were plated on selection plates with Kanamycin for both OmniMax 2 T1^R and TOP10 cells, and with 1 mM IPTG for just the OmniMAX 2 T1^R cells. Ten to twelve transformants were analyzed by colony PCR.

Helpful Hint

Plate OmniMAX 2 T1^R cells with 1 mM IPTG added to the plating medium to reduce background from empty vector, therefore facilitating screening and selection.

High stain sensitivity

Invitrogen™ SYBR™ Safe DNA Gel Stain is the recommended gel stain. It offers the highest sensitivity by utilizing blue light, eliminating the damaging effects of UV light exposure (Figure 10). While ethidium bromide (EtBr) is still a sensitive stain, the use of UV light can nick DNA and reduce cloning efficiency. Crystal violet stain utilizes white light and therefore does not damage the DNA; however, it is less sensitive.