The tube containing the TOPO™ TA vector arrived yellow instead of the usual red color. What does this indicate? Will this color change affect the activity of the Topoisomerase?

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Answer

Phenol red is added to the TOPO™ vector (pCRII, pCR2.1, and pCR4), mostly to make it more visible in the tube. The color of the solution is generally pink at room temperature, but at a low pH phenol red will turn yellow. We have tested acidic conditions (low pH) for the TOPO™ ligation reaction and found that the activity is not affected. At a pH 2.0, there was no observed decrease in efficiency. However, if the TOPO™ vector turns dark red, or is blue after adding the PCR product, then the PCR product buffer is too basic (pH 9). High pH does have a negative effect on the TOPO™ vectors, and the number of resulting colonies in this case will drop.

Note: The Directional TOPO™ and Zero Blunt™ TOPO™ vectors contain bromophenol blue dye rather than phenol red, which results in a blue-colored solution. Consult the product manual to confirm which dye is added to your vector, or contact Technical Support for more information.

Answer Id: E3341

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I’m getting very few colonies after transformation of my TOPO™ cloning reaction. How can I increase the number of primary colonies?

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Please try the suggestions below to increase the number of colonies.
- Longer incubation of the TOPO™ cloning reaction at room temperature, provided that the 6X Salt solution is added to the reaction.
- Electroporation can 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:
- Add 100 μL double diH2O to the 6 μL TOPO™ reaction and incubate 10 more minutes at 37 degrees C.
- Precipitate by adding 10 μL 3 M Na-Acetate, 2 μL 20 μg/μL glycogen, 300 μL 100% ethanol. Place on dry ice or –80 degrees C for 20 min, spin at top speed in a microcentrifuge at 4 degrees C 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).
- 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 mins incubation is not absolutely necessary, but it sufficiently dilutes the reaction and may help inactivate topoisomerase so that it is more easily electroporated.

Answer Id: E6761

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What can inhibit the TOPO™ cloning 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., to 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.

Answer Id: E6747

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I’m getting low to no colonies after transformation. Why?

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Some possible causes and remedies are:
- Ligase function is poor. Check the age of the ligase and function of the buffer.
- Competent cells are not transforming. Test the efficiency of the cells with a control supercoiled vector, such as puc19.
- Both molecules were de-phosphorylated.
- Inhibition of ligation by restriction enzymes and residual buffer. Try transformation of uncut vector, clean up restriction with phenol, or carry out PCR cleanup/gel extraction before ligation.
- Incorrect antibiotic selection used. Check the plasmid and plates and make sure concentration of antibiotic used is correct.

If nothing above applies, low to no colonies may be due to instability of the insert DNA in your competent cells. In this case, E. coli strains such as Stbl2™, Stbl3™, or Stbl4™ have been shown to support the propagation of DNA with multiple repeats, retroviral sequences, and DNA with high GC content better than other strains.

Answer Id: E6667

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I’m getting low cloning efficiency with my TOPO™ cloning reactions. What should I do?

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Please consider the following possible causes:
- pH > 9: Check the pH of the PCR amplification reaction and adjust with 1 M Tris-HCl, pH 8.
- Excess (or overly dilute) PCR product: Reduce (or concentrate) the amount of PCR product.
- Incomplete extension during PCR: Be sure to include a final extension step of 7 to 30 minutes during PCR. Longer PCR products will need a longer extension time.
- Cloning large inserts (>1 kb): Try one or all of the following suggestions: Increase amount of insert. Incubate the TOPO™ ™ cloning reaction longer. Gel-purify the insert using either a silica-based DNA purification system (e.g., PureLink™ system) or electroelution. Be sure that all solutions are free of nucleases (avoid communal ethidium bromide baths, for example.)
- PCR product does not contain sufficient 3’ A-overhangs even though you used Taq polymerase: Increase the final extension time to ensure all 3’ ends are adenylated. Taq polymerase is less efficient at adding a nontemplate 3’ A next to another A. Taq is most efficient at adding a nontemplate 3’ A next to a C. You may have to redesign your primers so that they contain a 5’ G instead of a 5´ T.

Answer Id: E6763

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What is the difference between a stop solution and salt solution? What is its function in the TOPO™ kit?

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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.

Answer Id: E6748

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I’m trying to decide between your pCR2.1 TOPO™ and pCR4-TOPO™ vectors to clone a 150 bp PCR product for sequencing. Which would you recommend?

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Answer

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.

Answer Id: E6754

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I typically store my PCR products before TOPO™ cloning. Is this okay?

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

Answer Id: E6744

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How does blue/white screening work?

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Answer

If working with a vector that contains the lac promoter and the LacZ alpha fragment (for ? 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 >400 bp. 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 β-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 mg/mL and IPTG to 0.1 mM to the medium. When adding directly on the surface of the plate, add 40 μl X-gal (20 mg/mL stock) and 4 μl IPTG (200 mg/mL stock).

Answer Id: E6664

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How do I adapt my cloning vector for TOPO™ cloning?

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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.

Answer Id: E6741

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The clones I’m selecting show deleted inserts. Why?

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Answer

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, retroviral sequences, and DNA with high GC content better than other strains.

Answer Id: E6668

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I’m getting no colonies after transformation. What should I do?

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Answer

No colonies may occur due to the following problems:

Bacteria were not competent. Use the pUC18 vector included with the One Shot™ module to check the transformation efficiency of the cells.
- Incorrect concentration of antibiotic on plates, or the plates are too old. Use 100 μg/mL of ampicillin or 50 μg/mL kanamycin. Be sure ampicillin plates are fresh (< 1 month old).
- The product was phosphorylated (TOPO™ cloning only). 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. The TOPO™ vector has a 3’ phosphate to which topoisomerase is covalently bound and a 5’ phosphate. The non- TOPO™ vectors (TA and Blunt) have a 3’ OH and a 5’ phosphate. Phosphorylated products should be phosphatased (CIP) before TOPO-cloning.

Answer Id: E6764

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Can I store my TOPO™ vector plus insert reaction? At what temperature?

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Answer

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.

Answer Id: E6749

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How does TA Cloning™ work?

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Answer

Taq polymerase has a non-template-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3´ ends of PCR products. The linearized vector supplied in our TA Cloning™ kits have single, overhanging 3´ deoxythymidine (T) residues. This allows PCR inserts to ligate efficiently with the vector.

Answer Id: E4061

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Do I need to gel purify my PCR product for TOPO™ cloning?

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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.

Answer Id: E6745

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