How does the protocol used in the TOPO XL-2 kit improve the efficiency of cloning long PCR products?
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.
What are the insert size limitations of TOPO cloning kits?
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 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.
Are there suggestions to increase the efficiency of cloning a long PCR insert into a TOPO vector (other than using TOPO XL-2 PCR cloning kit)?
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.
How do I incorporate additional restriction sites into my PCR primers to be able to restriction digest the insert after cloning into a TOPO vector?
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 Invitrogen Anza Restriction Enzyme Cloning System has a table in Appendix B of the User Guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/Anza_man.pdf) 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 that the cloning destination vector has compatible ends.
Additional primer design tips can be found here: http://www.thermofisher.com/us/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/primer-design-tools.html
I would like to clone my PCR product into a TOPO vector. When should I use kanamycin antibiotic for selection instead of ampicillin?
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 genes PCR amplified from vectors containing one of the antibiotic resistant genes (for instance ampicillin), it is best to select with the alternate antibiotic or both (kanamycin or kanamycin and ampicillin).
Should I use the gel extraction or the PCR purification method before cloning inserts into the pCR-XL-2-TOPO vector?
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. However, 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.
How can SYBR Safe DNA Gel Stain help my cloning efficiency with the pCR-XL-2-TOPO vector?
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 visualizing 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 at www.thermofisher.com/safeimager.
Can I use the TOPO XL-2 Complete PCR Cloning Kit to clone a 1 kb or smaller PCR fragment?
The TOPO XL-2 Complete PCR Cloning Kit has been tested with fragments as small as 1 kb and found to be extremely efficient.
What should I know about insert to vector molar ratio for the TOPO XL-2 Complete PCR Cloning Kit?
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.
For cloning into the pCR-XL-2-TOPO vector, how do I incorporate additional restriction sites into my primers?
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 Invitrogen Anza Restriction Enzyme Cloning System has a table in Appendix B of the User Guide (https://tools.thermofisher.com/content/sfs/manuals/Anza_man.pdf) 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: http://www.thermofisher.com/us/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/primer-design-tools.html
I am using the TOPO XL-2 Complete PCR Cloning Kit. When should I use kanamycin antibiotic for selection instead of ampicillin?
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).
I am using the TOPO XL-2 Complete PCR Cloning Kit. Should I use the gel extraction or the PCR purification method for purification of my insert?
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.
How can I improve my cloning efficiency with the pCR-XL-2-TOPO vector?
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 on the Thermo Fisher Scientific website at www.thermofisher.com/safeimager.