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Product FAQ

How does the pCR™4-TOPO™ and pCR™4Blunt-TOPO™ streamline sequencing?

Answer

Both the pCR™4-TOPO™ and pCR™4Blunt-TOPO™ vectors contain a minimized multiple cloning site. There are only 33 base pairs from the sequencing primer sites to the cloning site. This allows for more sequencing read of your insert and less of the vector which an save time analyzing the sequence data.

Answer Id: E3337

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Product FAQ

What are some tips you can give me to obtain the highest transformation efficiency?

Answer

Some suggestions that will help you to obtain the highest transformation efficiency are:
- Thaw competent cells on ice instead of room temperature; do not vortex cells.
- Add DNA to competent cells once thawed.
- Ensure that the incubation times are followed as outlined in the competent cell protocol for the strain you are working with; changes in the length of time can decrease efficiency.
- Remove salts and other contaminants from your DNA sample; DNA can be purified before transformation using a spin column, or phenol/chloroform extraction and ethanol precipitation can be employed.

Answer Id: E6709

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Product FAQ

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

Answer

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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Product FAQ

Why is there a TOPO™ Cloning kit "Stop Solution" mentioned in my manual, but it is not supplied in the TOPO™ Cloning kit?

Answer

Stop Solution is a reagent included in older versions of all TOPO™ kits, and still offered in the TOPO™ XL kits. It is very similar to the current TOPO™ cloning kit Salt Solution. Instruction manual versions preceding Version J recommended adding the Stop Solution at the end of the 5 minute TOPO™ reaction. The basic function of the Stop/Salt Solution is to keep the Topoisomerase from re-binding to the plasmid DNA and nicking the DNA. Later studies found that it was actually more effective to add this component during the TOPO™ cloning reaction versus afterward, and thus the Stop Solution was replaced with the similar but higher-concentration Salt Solution in most kits.

Answer Id: E3339

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Product FAQ

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

Answer

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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Product FAQ

I’m transforming pCR™2.1-TOPO™ clones into TOP10F’ cells. Will I need to add IPTG to my plates along with X-gal for blue/white screening? What if I used TOP10 cells instead?

Answer

The F’ episome in TOP10F’ has a lacIq marker, which over-expresses the lac repressor. IPTG must be added to LB plates along with X-gal to see beta-galactosidase expression and blue color in this strain. TOP10, on the other hand, does not require IPTG for blue/white screening.

Answer Id: E6727

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Product FAQ

What happens when a vector adapted with the Topoisomerase enzyme is stored at -80°C?

Answer

-80°C storage is fine. The TOPO™ vector can be freeze-thawed several times without loss of activity, and the vector is stable for storage at either -20°C or -80°C. However, note that the vector rapidly ages at room temperature (~15 min), which will result in a lower cloning efficiency (a reduction in the number of colonies following transformation). So during active use it should be kept on ice to prevent degradation, and should be quickly returned to the freezer.

Answer Id: E3340

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

Answer

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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Product FAQ

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

Answer

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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Product FAQ

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?

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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Product FAQ

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

Answer

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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Product FAQ

Can I transform 2 plasmids into the same cell?

Answer

Yes, this is possible. We recommend using saturating amounts of DNA (10 ng of each plasmid). Make sure that the origin of replication is different in each plasmid so that they can both be maintained in the cell. If the ori is the same, the plasmids will compete for replication and the one with even a slight disadvantage will be lost. Alternatively, cells with a resident plasmid can be electroporated with a second plasmid without “electrocuring” taking place.

Answer Id: E6712

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Product FAQ

I’m trying to clone a gene that has multiple repeated sequences into my pCR™2.1-TOPO™ vector, followed by transformation into TOP10 cells. My clones contain random rearrangements and deletions. What can I do?

Answer

With any strain, the first thing to try would be to lower the growth temperature of the culture to 40 degrees C or even lower (room temperature). Slower growth will generally allow E. coli to tolerate difficult sequences better. If reducing the growth temperature doesn’t help, you may want to consider using a competent cell strain such as Stbl2™ or Stbl4™ cells, which have been shown to accommodate this type of sequence better than other strains in the same conditions.

Answer Id: E6729

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Product FAQ

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?

Answer

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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