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K412501

Product FAQ

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

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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Product FAQ

What are some of the prerequisites for TOPO™ cloning?

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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Product FAQ

I’m trying to transform large plasmid, 40 kb in size. What strain should I use?

Answer

While there is no specific strain that works better with large plasmids, it is important to focus on transformation efficiency. For larger plasmids, chemically competent cells with highest efficiency are suggested, such as OmniMAX™ 2, TOP10, etc. We would recommend using an electrocompetent cell strain with plasmids larger than 20 kb for best efficiency.

Answer Id: E6730

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Product FAQ

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

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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Product FAQ

How should I store my competent E. coli?

Answer

We recommend storing our competent E. coli strains at -80°C. Storage at warmer temperatures, even for a brief period of time, will significantly decrease transformation efficiency.

Answer Id: E9874

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Product FAQ

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

Answer

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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Product FAQ

What is the best ratio of insert:vector to use for cloning? Is there an equation to calculate this?

Answer

You may have to try different ratios ranging from 1:1 to 15:1 insert:vector.

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: E7645

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Product FAQ

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

Answer

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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Product FAQ

Can I store my competent E. coli in liquid nitrogen?

Answer

We do not recommend storing competent E. coli strains in liquid nitrogen as the extreme temperature can be harmful to the cells. Also, the plastic storage vials are not intended to withstand the extreme temperature and may crack or break.

Answer Id: E9875

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Product FAQ

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

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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Product FAQ

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?

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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Product FAQ

What concentrations do you typically recommend for X-gal and IPTG for blue/white screening?

Answer

In plates, we recommend 50 μg/mL X-gal and 1 mM IPTG (0.24 mg/mL). When spreading directly onto agar plates, we recommend 40-50 μl of 40 mg/mL X-gal (2% stock) in dimethylformamide and 30-40 μl of 100 mM IPTG on top of the agar. Let the X-gal and IPTG diffuse into the agar for approximately 1 hour. Do not plate on media containing glucose, as it competes with X-gal or bluo-gal and prevents cells from turning blue.

Answer Id: E6711

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Product FAQ

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?

Answer

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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Product FAQ

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

Answer

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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Product FAQ

Can I run the TOPO™ vector on a gel?

Answer

No, we do not recommend this as these vectors contain the topoisomerase DNA protein complex conjugated to the end of the vector.

Answer Id: E6739

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