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

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Answer

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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I have a blunt-ended restriction product cut from another cloning vector. Can I insert this into a Zero Blunt™ TOPO™ vector?

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

Answer Id: E6755

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

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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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I’m able to get a lot of colonies, however, none contain my insert of interest. What should I do?

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You may be cloning in an artifact. TA and TOPO™ Cloning are very efficient for small fragments (< 100 bp) present in certain PCR reactions. Gel-purify your PCR product using either a silica-based DNA purification system or electroelution. Be sure that all solutions are free of nucleases (avoid communal ethidium bromide baths, for example.)

Answer Id: E6766

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I’m seeing a lot of vector-only colonies when I try to perform a negative control reaction using vector only (no insert) for a TOPO™ reaction. Is my TOPO™ vector re-ligating?

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Using the vector only for transformation is not a recommended negative control. The process of topo-adaptation is not a 100% process, therefore, there will be “vector only” present in your mix, and colonies will be obtained.

Answer Id: E6768

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I’m planning on cloning a 1kb fragment for sequencing and want to minimize the amount of vector sequence in my data. Which of your vectors should I use?

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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 pCR4Blunt-TOPO™ vector.

Answer Id: E6756

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What is the difference between the pCR™2.1-TOPO™ and pCR™4-TOPO™ vectors?

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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.

Answer Id: E9851

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What PCR enzyme would you recommend for use with the Directional TOPO™ Cloning Kits?

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For the Directional TOPO™ Cloning Vectors, a PCR product must be generated by a proofreading enzyme to create a blunt product. Pfx50™ or Accuprime™ Pfx and Accuprime™ Pfx Supermix from Life Technologies are recommended for use.

When cloning a Pfx-amplified PCR product, the insert to vector ratio is an important consideration. The PCR product generally needs to be diluted since Pfx generates a high concentration of product and using too much insert DNA can hamper the TOPO™ reaction. A 1:1 molar ratio of vector to insert (or about 2-10ng of insert) is recommended.

Answer Id: E4290

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A majority of colonies are blue or light blue, with very few white colonies. What should I do?

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Answer

There could be a few possibilities for this:

- The insert does not interrupt the reading frame of the lacZ gene. If you have a small insert (< 500 bp), you may have light blue colonies. Analyze some of these blue colonies as they may contain insert.
- A polymerase that does not add 3’ A-overhangs was used. If you use a proofreading enzyme such as AccuPrime™ Pfx or Platinum™ Pfx™, you will need to do a post-reaction treatment with Taq to add the 3’ A-overhangs.
- PCR products were gel-purified before ligation. Gel purification can remove the single 3’ A- overhangs. Otherwise, optimization of your PCR can be performed so that you can go directly from PCR to cloning.
- The PCR products were stored for a long period of time before ligation reaction. Use fresh PCR products. Efficiencies are reduced after as little as 1 day of storage.
- Too much of the amplification reaction was added to the ligation. The high salt content of PCR can inhibit ligation. Use no more than 2-3 μl of the PCR mixture in the ligation reaction.
- The molar ratio of vector:insert in the ligation reaction may be incorrect. Estimate the concentration of the PCR product. Set up the ligation reaction with a 1:1 or 1:3 vector:insert molar ratio.
On a typical plate there are a few white colonies which do not contain insert. These 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). To find a colony with an insert it is best to pick clones of a variety of color and pattern for analysis. Often an insert will generate two distinct patterns according to its orientation.

Answer Id: E6765

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What is the best molar ratio of PCR product:vector to use for TOPO™ TA cloning? Is there an equation to calculate the quantity to use?

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We suggest starting with a molar ratio of 1:1 (insert:vector), with a range of 0.5:1 to 2:1. The quantity used in a TOPO™ cloning reaction is typically 5-10 ng of a 2 kb PCR product.

Equation:

length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 (insert:vector ratio)

Answer Id: E7648

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I’m trying to clone in my phosphorylated PCR product into a TOPO™ vector, and I’m getting no colonies. However, when I clone the same product into a TA vector, everything works perfectly. Why is this?

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Answer

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 phosphatased (CIP) before TOPO™ cloning.

Answer Id: E6767

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Can I use the pCR™-Blunt or pCR-Blunt-TOPO™ vector to clone PCR products amplified with Taq DNA polymerase?

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No you cannot use pCR™-Blunt or pCR-Blunt-TOPO™ vector to clone PCR products amplified with Taq DNA polymerase. The pCR-Blunt vector is prepared with blunt ends to accept blunt-ended fragments. Due to the terminal transferase activity of Taq DNA polymerase, PCR products amplified with this enzyme have 3’-A overhangs. In order to clone these products into pCR™-Blunt, you would need to polish the ends to make them blunt (which usually is not an efficient process). Our TA Cloning™ Kits or TOPO™ TA Cloning™ kits are a better choice for cloning Taq-generated PCR products. TA Cloning™ kits include a linearized vector with 3’-T overhangs for efficient ligation of Taq-generated PCR products without additional manipulation.

Answer Id: E4063

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What is the difference between the pCR™2.1-TOPO™ and pCR™II-TOPO™ vectors?

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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.

Answer Id: E9850

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Which PCR polymerases do you recommend for TA/Blunt/D-TOPO cloning and why?

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TA Cloning:
- This cloning method was designed for use with pure Taq polymerases (native, recombinant, hot start); however, High Fidelity or Taq blends generally work well with TA cloning. A 10:1 or 15:1 ratio of Taq to proofreader polymerase will still generate enough 3' A overhangs for TA cloning.
- Recommended polymerases include Platinum™ Taq, Accuprime™ Taq, Platinum™ or Accuprime™ Taq High Fidelity, AmpliTaq™, AmpliTaq Gold™, or AmpliTaq Gold™ 360.

Blunt cloning:
- Use a proofreading enzyme such as Platinum™ SuperFi™ DNA Polymerase.

Directional TOPO cloning:
- Platinum™ SuperFi™ DNA Polymerase works well.

Answer Id: E6651

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