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K250020

Product FAQ

Your Gateway™-adapted TOPO™ vectors are supplied with a control template and control primers. Can I obtain the sequence of the control template?

Answer

The sequence of the control template is proprietary.

Answer Id: E9853

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Product FAQ

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?

Answer

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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Product FAQ

How does TA Cloning™ work?

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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Product FAQ

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

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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Product FAQ

How does blue/white screening work?

Answer

If working with a vector that contains the lac promoter and the LacZ alpha fragment (for ? 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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Product FAQ

Can I order my TOPO™ vector as a standalone product? I have plenty of competent cells.

Answer

Yes, our pCR.1 TOPO™ TA (Cat. No. 450641), pCR4-TOPO™ TA (Cat. No. 450030), pCRBluntII-TOPO™ (Cat. No. 450245) are available separately.

Answer Id: E6740

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Product FAQ

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?

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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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

I’m getting low cloning efficiency with my TOPO™ cloning reactions. What should I do?

Answer

Please consider the following possible causes:
- pH > 9: Check the pH of the PCR amplification reaction and adjust with 1 M Tris-HCl, pH 8.
- Excess (or overly dilute) PCR product: Reduce (or concentrate) the amount of PCR product.
- Incomplete extension during PCR: Be sure to include a final extension step of 7 to 30 minutes during PCR. Longer PCR products will need a longer extension time.
- Cloning large inserts (>1 kb): Try one or all of the following suggestions: Increase amount of insert. Incubate the TOPO™ ™ cloning reaction longer. Gel-purify the insert using either a silica-based DNA purification system (e.g., PureLink™ system) or electroelution. Be sure that all solutions are free of nucleases (avoid communal ethidium bromide baths, for example.)
- PCR product does not contain sufficient 3’ A-overhangs even though you used Taq polymerase: Increase the final extension time to ensure all 3’ ends are adenylated. Taq polymerase is less efficient at adding a nontemplate 3’ A next to another A. Taq is most efficient at adding a nontemplate 3’ A next to a C. You may have to redesign your primers so that they contain a 5’ G instead of a 5´ T.

Answer Id: E6763

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Product FAQ

How does ccdB selection work?

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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Product FAQ

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

Answer

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

Answer Id: E4398

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Product FAQ

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

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 Thermo Fisher Scientific 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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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

Can you go directly from a pENTR™/D-TOPO™ reaction into an LR Clonase™ Reaction without first purifying the DNA?

Answer

In most cases there will not be enough pENTR™ vector DNA present to go directly from TOPO™ cloning into an LR reaction. You need between 100-300 ng of pENTR™ vector for an efficient LR reaction, and miniprep of a colony from the TOPO™ transformation is necessary to obtain that much DNA. However, if you want to try it, here are some recommendations for attempting to go straight into LR reactions from the TOPO™ reaction using pENTR™/D, or SD TOPO™, or pCR™8/GW/TOPO™ vectors:

1. Heat inactivate the topoisomerase after the TOPO™ cloning reaction by incubating the reaction at 85C for 15 minutes.
2. Use the entire reaction (6 uL) in the LR clonase reaction. No purification steps are necessary.
3. Divide the completed LR reaction into 4 tubes and carry out transformations with each tube. You cannot transform entire 20 uL reaction in one transformation, and we have not tried ethanol precipitation and then a single transformation.

When attempting this protocol, we observed very low efficiencies (~10 colonies/plate). So just be aware that while technically possible, going directly into an LR reaction from a TOPO™ reaction is very inefficient and will result in a very low colony number, if any at all.

Answer Id: E3953

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Product FAQ

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

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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