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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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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I’ve cloned my gene into the pCR™2.1-TOPO™ vector and transformed into the TOP10 cells that came with the kit. I then did a plasmid miniprep followed by digestion of the DNA with XbaI. However, the vector is not cutting correctly. What happened?

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XbaI cutting site is a Dam-methylation sensitive restriction site. TOP10 is a dam(+) strain, which means it expresses the methylating enzyme, Dam. You can try re-transforming into a dam(-) strain, such as INV110. Other dam- (and dcm-) sensitive restriction sites include the following:

- Dam: Bcl I, Cla I, Hph I, Mbo I, Mbo II, Taq I, Xba I, BspH I, Nde II, Nru I
- Dcm: Ava II, EcoO 109 I, EcoR II, Sau96 I, ScrF, Stu I, Aat I, Apa I, Bal I, Kpn I, ISfi I

Answer Id: E6724

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What considerations should I take into account when designing primers for PCR of an insert which will be cloned into a TOPO™ vector?

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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.

Answer Id: E6746

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I’m subcloning fragments of yeast genomic DNA into a TOPO™ vector. I’m seeing a lot of deletions in the clones I’m selecting. What can I do?

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Answer

If you are using a mcr/mrr(+) competent cell strain, cellular enzymes may be recognizing eukaryotic methylation patterns on the yeast genomic DNA and deleting or rearranging it. Try a mcr/mr(-) strain such as Top10, DH10B™, or OmniMAX™ 2.

Answer Id: E6725

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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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I’m getting no colonies at all on my plates. Can you help?

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We recommend trying the following:
- Carry out the puc19 transformation control; this gives you information about the performance of the cells.
- Check plates for expiration and correct media used (LB/agar).
- Confirm that the correct antibiotic and concentration was used.

Answer Id: E6728

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How is competent cell efficiency measured? How is it calculated?

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Competent cell efficiency is measured by transformation efficiency. Transformation efficiency is equal to the number of transformants, or colony forming units, per microgram of plasmid DNA (cfu/microgram).

Answer Id: E6710

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How does blue/white screening work?

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If working with a vector that contains the lac promoter and the LacZ alpha fragment (for α 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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What can inhibit the TOPO™ cloning reaction?

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Answer

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 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’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 plating my untransformed TOP10 cells on ampicillin as a negative control, but see a lot of colonies on the plates.

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There are a few conditions that can lead to this: SOC medium or other media used when plating was contaminated, DNA was contaminated with amp-resistant microbes, you used old plates with degraded amp, or the competent cells themselves were contaminated.

Answer Id: E6726

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Can I use a DNA polymerase mixture containing both Taq polymerase and a proofreading polymerase for TA Cloning™?

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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.

Answer Id: E4062

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Where can I obtain Spectinomycin for use with your pCR™8/GW/TOPO™ vector?

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Answer

Spectinomycin dihydrochloride is available from Sigma (Catalog no. S4014).   

Answer Id: E4398

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