I’m only getting white colonies, but none of the clones have an insert. What can I do?

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Answer

Here are a few suggestions:

- Small fragments/linkers are cloning in to your vector instead of your insert; to correct this, gel-purify the insert before ligation
- Ensure that the correct concentrations of X-gal and/or IPTG (if vector contains the lacIq marker) are used
- If spreading X-gal and/or IPTG on your plate, allow sufficient time for the reagents to diffuse into the plate
- Incubate your plate for a longer period to ensure full color development

Answer Id: E6732

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I’m getting overgrowth of colonies. Why?

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Answer

Ensure that you are using the correct antibiotic at the appropriate concentration. Additionally, make sure the antibiotic is not expired. If colonies exhibit unexpected morphologies, contamination could be a factor. Check your S.O.C. medium and LB growth medium.

Answer Id: E6733

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I have a TOPO TA Cloning™ kit with TOP10 cells. I ran out of competent cells but still have vector left. I also have subcloning DH5α™ cells and TOP10F’ cells in the freezer. Are either of these cells compatible? What strain features should I be aware of?

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Answer

Subcloning DH5α™ cells are a compatible strain, but you will get lower efficiency (10e6 vs 10e9) and therefore risk getting fewer clones. Top10F’ is also compatible, but if blue/white screening is performed, IPTG along with X-gal will be needed for detection due to the expression of the lacIq repressor present in cells containing an F’ episome.

Answer Id: E6734

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What are the insert size limitations of TOPO™ cloning kits?

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Answer

Regular TOPO™ TA Cloning™ kits are efficient for cloning PCR products up to approximately 2-3kb. With PCR products larger than 3kb, the efficiency of cloning drops significantly. The TOPO™ XL PCR Cloning kit has been optimized for TOPO™ cloning of long (3-10kb) 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 20μl, while maintaining same amount of vector and insert. Increase the volume of the salt solution to 3.7μl to compensate for the increase in volume. Diluting the reaction reduces the competition for the vector ends.

Answer Id: E6751

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

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Answer

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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I received my TOPO™ vector and the solution is colored. Is it okay to use?

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Answer

TOPO™ and TOPO™ TA vectors (non-directional) have phenol red dye added. The color should be pink (or yellow) at room temperature. If it turns blue when PCR product is added, the PCR product buffer is too basic and the number of transformed colonies will drop. When the solution is yellow, it signifies an acidic pH. At a pH 2.0, TOPO™ vectors still maintain high cloning efficiency. Directional TOPO™ and Zero Blunt™ TOPO™ vectors have bromophenol blue dye added.

Answer Id: E6735

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

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Answer

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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What are some of the prerequisites for TOPO™ cloning?

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Answer

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.

Answer Id: E6736

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What are the advantages of using a TOPO™ TA cloning system compared to traditional TA cloning?

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Answer

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.

Answer Id: E6742

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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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What range of PCR product (molar ratios and ng quantities) do you suggest for TOPO™ TA cloning?

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Answer

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

Answer Id: E6737

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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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Why does the pCR8/GW/TOPO™ vector contain spectinomycin resistance? Do you offer spectinomycin?

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Answer

The pCR8/GW/TOPO™ entry vector uses spectinomycin for selection so that the Entry clone that is generated can be used with any Destination vector. Most Destination vectors have ampicillin resistance although there are a few that have kanamycin or zeocin resistance. Otherwise, the background will be too high, unless the Entry vector is first linearized. Spectinomycin is a less commonly used selectable marker. We do not offer spectinomycin; spectinomycin dihydrochloride is available from Sigma (Cat. No. S4014).

Answer Id: E6813

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I’m able to see colonies on a plate, but when I pick them for liquid culture, no growth is observed. Why?

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Answer

One possible explanation could be toxicity associated with the insert. This toxicity does not affect slow growing cells on solid medium but is much stronger in faster growth conditions like liquid medium.

Suggestions:
1. Use TOP10F’ or any other strain with the LacIq repressor
2. Try using any other strain appropriate for cloning.
3. Lower growth temperature to 27 - 30 degrees C and grow the culture longer
4. Another possibility to explain lack of growth is possible phage contamination. In this situation we recommend using an E. coli strain that is T1 phage resistant like DH5alpha-T1R.

Answer Id: E6669

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I’m trying to decide between the TOP10, DH5α™, and Mach1™ strains you have for my TOPO™ TA Cloning™ reactions. Can you explain the significant differences between these strains?

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Answer

DH5α™ cells are commonly used for routine cloning, but are mcr/mrr+, and therefore not recommended for genomic cloning. The TOP10 competent cells, on the other hand, contain mutated mcr/mrr, and therefore are a good choice for routine cloning and can be used for cloning of methylated DNA, such as eukaryotic genomic DNA. Our Mach1™ strain is the fastest growing cloning strain that is T1 phage resistant.

Answer Id: E6704

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