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

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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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3a5aa414a7857e9951a95203bc5bbf09_FAQ

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

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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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38250f5978487e893b93e370635ae734_FAQ

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

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-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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78b0fa3aceaefd3ca5241fe09ef4cf5a_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?

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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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93c71b325e3d330fec2eb9f7714a33c8_FAQ

1. Can  the TOPO TA cloning kits (TOPO TA for sequencing, Zero blunt, Zero Blunt TOPO, Original TA cloning kit, Dual Promoter cloning kit) be used for in vitro transcription to generate probes? 2. Can they be used to do in vitro translation?

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Answer

1. Yes, the TOPO® TA Cloning® and Zero Blunt® cloning vectors can be used for in vitro transcription. The T7, SP6, and T3 promoters contained in these vectors are active phage promoters, and the corresponding polymerases will transcribe RNA from these promoters.

2. No, these vectors are not generally recommended for in vitro translation because most of these vectors contain one or more translational initiation signals (ATG) downstream (3') of the promoters contained in them, within the multiple cloning sites, which may interfere with attempts to translate an open reading frame in the insert.

Answer Id: E3945

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0f6f69ea291373a65a0a3645a3e7ff0b_FAQ

What are the melting temperatures for the M13 Forward (-20) and M13 Reverse primers in the TOPO® Cloning and Zero Blunt® Kits?

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Answer

Assuming that the primer is at a 50 nM final concentration and 50 mM final salt concentration, the melting temperatures are: M13 Forward (-20) Primer = 52.7 and the M13 Reverse Primer = 45.3. For use in the control PCR reaction we recommend using an annealing temperature of 56C.

Answer Id: E4024

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815dae5afa8bbcc366587e4d3d919944_FAQ

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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fb53c1ece88718be1d4da1509411423b_FAQ

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

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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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340dfd4c1b12d5bd85bdb219d563375d_FAQ

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

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Answer

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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337cdf091765fed4a7afa0de58e0d347_FAQ

Does Platinum® Taq DNA Polymerase High Fidelity enzyme mix leave 3’ A-overhangs on the PCR product for subsequent cloning into a TOPO® TA or original TA vector?

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Answer

Yes, the enzyme mix leaves 3’ A-overhangs on a portion of the PCR products. However, the cloning efficiency is greatly decreased compared to that obtained with Taq polymerase alone. It is recommended to add 3’ A-overhangs to the product for TA cloning.

Answer Id: E7268

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d878f3e417a6e80a3b509244537a92c6_FAQ

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

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Answer

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

B. Blunt cloning:
- Use proofreading enzymes such as Pfx50, Platinum® or AccuPrime™ Pfx.

C. Directional TOPO cloning:
- Pfx50 or AccuPrime Pfx work well.

Answer Id: E6651

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c282cdd6154484d04bb8b2ce3e2275db_FAQ

Can I use TOPO®TA pCR2.1 or pCR II or pCR4 for my protein expression experiments?

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Answer

No, these vectors do not contain a functional promoter to express your gene of interest. These vectors are typically for subcloning or sequencing.

Answer Id: E6661

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68e8de1e920dec09a9ab5dd783827991_FAQ

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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467e1a97574e4e4d9e5ce269a7d4d16d_FAQ

How does ccdB selection work?

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Answer

TOPO® vectors containing the LacZ-ccdB cassette allow direct selection of recombinants via disruption of the lethal E. coli gene, ccdB. Ligation of a PCR product disrupts expression of the LacZ-ccdB gene fusion permitting growth of only positive recombinants upon transformation. Cells that contain non-recombinant vector are killed upon plating. Therefore, blue/white screening is not required. When doing blue/white color screening of clones in TOPO® vectors containing the LacZ-ccdB cassette, colonies showing different shades of blue may be observed. It is our experience that those colonies that are light blue as well as those that are white generally contain inserts. The light blue is most likely due to some transcription initiation in the presence of the insert for the production of the lacZ alpha without enough ccdB expressed to kill the cells and is insert dependent. To completely interrupt the lacZ gene, inserts must be >400 bp; therefore an insert of 300 bp can produce a light blue colony. A white colony that does not contain an insert is generally due to a spontaneous mutation in the ccdB gene.
A minimum insertion of 150 bp is needed in order to ensure disruption of the ccdB gene and prevent cell death. (Reference: Bernard et al., 1994. Positive-selection vectors using the F plasmid ccdB killer gene. Gene 148: 71-74.) Strains that contain an F plasmid, such as TOP10F’, are not recommended for transformation and selection of recombinant clones in any TOPO® vector containing the ccdB gene. The F plasmid encodes the CcdA protein, which acts as an inhibitor of the CcdB gyrase-toxin protein. The ccdB gene is also found in the ccd (control of cell death) locus on the F plasmid. This locus contains two genes, ccdA and ccdB, which encode proteins of 72 and 101 amino acids respectively. The ccd locus participates in stable maintenance of F plasmid by post-segregational killing of cells that do not contain the F plasmid. The CcdB protein is a potent cell-killing protein when the CcdA protein does not inhibit its action.

Answer Id: E6665

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897dd8f2419e66e253ac03d76aea4d67_FAQ

How does selection with the LacZ gene work?

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Answer

If working with a vector that contains the lac promoter and the LacZ α 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.

Answer Id: E6666

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fbaab7c272d5a4edaa00f9a77bd23036_FAQ