PCR Cloning Support - Getting Started

Cloning efficiency of gel-purified products can be further increased by avoiding visualization of the bands with UV light. 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 DNA gel Stain and visualized with blue light on the Safe Imager 2.0 Blue-Light Transilluminator. Data showing improved cloning efficiency after gel purification with this method versus ethidium bromide and UV light is available here.

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. The TOPO XL-2 Complete PCR Cloning Kit is supplied with the PureLink Quick Gel Extraction and PCR Purification Combo Kit for added flexibility allowing for faster PCR purification when inserts are smaller, while primers and dNTPs still need to be removed.

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.

Both ampicillin and kanamycin are effective in selecting against growth of bacterial colonies that do not have recombinant clones (negative selection). However, it has been found that for genes that are PCR amplified from vectors containing one of the antibiotic-resistant markers (for instance ampicillin), it is best to select with the alternate antibiotic or both (kanamycin or kanamycin and ampicillin).

When designing PCR primers to produce amplicons with incorporated restriction sites, additional bases may be required in the regions flanking the restriction enzyme recognition site to allow complete digestion of the DNA fragment that is produced. The Anza Restriction Enzyme Cloning System has a table in Appendix B of the User Guide with a recommended number of additional bases needed to be added to either side of the recognition site to achieve complete digestion. If using restriction enzymes that leave cohesive or “sticky” ends, ensure the cloning destination vector has compatible ends. Additional primer design tips can be found here.

We recommend keeping the insert:vector molar ratio low, optimally 1:1, with 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.

The TOPO XL-2 Complete PCR Cloning Kit has been tested with fragments as small as 1 kb and found to be extremely efficient.

Yes, the TOPO® TA Cloning® and Zero Blunt® cloning vectors can be used for in vitro transcription. The T7, SP6, and T3 promoters contained in these vectors are active phage promoters, and the corresponding polymerases will transcribe RNA from these promoters.

These vectors are generally not recommended for in vitro translation because most of these vectors contain one or more translation initiation signals (ATG) downstream (3') of the promoters contained in them, within the multiple cloning sites, which may interfere with attempts to translate an open reading frame in the insert.

Please consider the following before TOPO® cloning:

• TOPO® cloning cannot ligate DNA with a 5’ phosphate group.
• TOPO® cloning will decrease in efficiency inversely with the size of the insert (above 3 kb) unless using the TOPO® XL cloning kit.
• TOPO® vectors contain different antibiotic resistance markers which should be considered before purchase.
• TOPO® TA vectors accept fragments containing a 3’ A overhang while Zero Blunt® vectors accept fragments that are blunt-ended.

Please consider the following when designing your primers:

• The 3’ pcr primer cannot contain homology to the 5’ flap sequence GTGG.
• The enzyme you use must create a blunt-ended PCR product for cloning.
• Primers cannot contain 5’ phosphates, which will block the 5’ OH nucleophile reactive group.
• The reading frame must be considered when you are designing your primers.

No, we do not recommend this as these vectors contain the topoisomerase DNA protein complex conjugated to the end of the vector.

Yes, our pCR™2.1 TOPO® TA (Cat. No. 450641), pCR™4-TOPO® TA (Cat. No. 450030), pCRBlunt®II-TOPO® (Cat. No. 450245) are available separately.

We offer a custom service for TOPO® cloning adaptation services. Our scientists can prepare your vector for either blunt TOPO® cloning, TOPO® TA cloning, or directional TOPO® cloning of PCR products.

The sequence of the control template is proprietary.

The vector backbones for both of these vectors are very similar. The main difference is that the pCR™II-TOPO® vector is a dual promoter vector, containing the SP6 and T7 promoters for in vitro transcription/sequencing, whereas the pCR™2.1-TOPO® vector contains only the T7 promoter for in vitro transcription/sequencing. Both vectors contain the M13 Forward and Reverse primer sites for sequencing or PCR screening.

The vector backbones for both of these vectors are very similar. The main difference is that the pCR™4-TOPO® vector has sequencing primer sites located as close as 33 base pairs from the PCR product insertion site. This minimizes the amount of vector DNA sequence that needs to be read before reaching the sequence of the insert, making the pCR™4-TOPO® vector very useful for sequencing applications.

TA cloning ligates the insert and vector using a T4 DNA ligase, while TOPO® TA cloning uses the intrinsic properties of topoisomerase, which ligates the insert and vector during a 5 minute desktop reaction. TOPO® TA cloning results in >95% recombinants, while TA cloning results in >80% recombinants.

If you wish to use a polymerase mixture containing Taq polymerase and a proofreading polymerase, Taq must be used in excess with a 10:1 ratio of Taq to the proofreading enzyme to ensure the presence of 3´ A-overhangs on the PCR product. If you use polymerase mixtures that do not have enough Taq polymerase or a proofreading polymerase only, you can add 3' A-overhangs following PCR. See the vector product manuals for details.

Some examples of Taq blends that are compatible with TOPO® TA cloning are Platinum® Taq DNA Polymerase High Fidelity and AccuPrime™ Taq DNA Polymerase High Fidelity.

No, your gene of interest must be amplified with a proofreading polymerase such as Platinum SuperFi DNA Polymerase or AccuPrime Pfx DNA Polymerase that leaves blunt ends for directional TOPO cloning.

For optimal TOPO® cloning, we recommend using fresh PCR products.

Gel purification is not required if the gel indicates that the PCR product is clean with no visible non-specific bands or primer dimers. 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. 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.

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. This is because the necessary phosphate group is contained within the topoisomerase-DNA intermediate complex of the vector. TOPO® vectors have a 3' phosphate to which topoisomerase is covalently bound and a 5' phosphate. Non-TOPO® linear vectors (TA and Blunt) have a 3' OH and a 5' phosphate. Phosphorylated products should be treated with phosphatase (CIAP) prior to TOPO-cloning. Treatment with CIAP may raise efficiency to 25%. 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.

The optimal ratio is 1:1 insert to vector. Optimization can be done using a ratio of 0.5-2 molecules of insert for every molecule of the vector.

Equation:

The composition of the 6X Stop solution is 0.3 M NaCl, 0.06 M MgCl2, and the composition of the 6X Salt solution is 1.2 M NaCl, 0.06 M MgCl2. Stop solution is only included in the TOPO® XL Cloning kit whereas Salt solution is currently included in all of the other TOPO® cloning kits. These solutions 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 4 degrees C 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 4 degrees C showed little to no decrease in the number of colonies when compared to fresh TOPO® reaction mix.

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 µL of a 10 mg/mL stock of EtBr per 100 mL of agarose gel is sufficient.
2. Increase incubation time of the TOPO® reaction to 30 minutes to an hour. 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 (range 0.5 to 1 or 2 to 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.

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.

Regular TOPO® TA Cloning® kits are efficient for cloning PCR products up to approximately 2-3 kb. With PCR products larger than 3 kb, the efficiency of cloning drops significantly. The TOPO® XL PCR cloning kit has been optimized for TOPO® cloning of long (3-10 kb) PCR products.

If using the regular TOPO® kits, here are some tips to improve efficiency:

1. Use crystal violet instead of ethidium bromide (EtBr) to visualize the PCR for gel isolation to avoid DNA nicks.

2. Increase incubation time of the TOPO® reaction to 30 mins.

3. Keep insert:vector molar ratio low, optimally 1:1.

4. Dilute 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.

The protocol includes a novel 15-minute gel purification step to improve the efficiency of cloning long PCR products. Gel purification eliminates smaller PCR products which preferentially ligate into the vector. In addition, the TOPO® XL PCR cloning kit protocol eliminates the use of ethidium bromide and UV light which can nick long PCR products and reduce cloning efficiency. The kit uses the non-toxic crystal violet reagent to allow visualization of DNA under ambient light. 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 and UV light visualization.

dATP is a competitive inhibitor. Phosphate will reduce ligation efficiency. Detergents in your ligation buffer will likely not affect activity. High levels (0.2 M) of Na2+, K+, Cs+, Li+, and NH4+ inhibit the enzyme almost completely. Polyamines, spermine, and spermidine also serve as inhibitors.

Due to the small size of your product, we recommend using the pCR™2.1 TOPO® vector for your cloning. This size fragment would not be able to fully interrupt the ccdB gene in the pCR4™-TOPO® vector, and therefore, you may not get colonies as ccdB is lethal to E. coli.

Yes, though you will need to treat it with calf intestinal phosphatase (CIP) to get rid of the 5’ phosphate group for TOPO® cloning.

We would suggest using our TOPO® TA cloning kit for sequencing, which contains the pCR™4 TOPO® vector, or our Zero Blunt® TOPO® PCR cloning kit for sequencing, which contains the pCR™4Blunt-TOPO® vector.

We would suggest using one of our pENTR D-TOPO® kits for easy TOPO® cloning of your insert with the advantage of directionality.

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.)

TOPO® vectors containing the LacZ-ccdB cassette allow direct selection of recombinants via disruption of the lethal E. coli gene, ccdB. Ligation of a PCR product disrupts expression of the LacZ-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.