Are there common restriction sites that can be used to excise a gene out of a Gateway® plasmid?

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Answer

The core region of the att sites contains the recognition sequence for the restriction enzyme BsrGI. Provided there are no BsrGI sites in the insert, this enzyme can be used to excise the full gene from most Gateway® plasmids. The BsrGI recognition site is 5'-TGTACA and is found in both att sites flanking the insertion site.
If a different restriction site is desired, the appropriate sequence should be incorporated into your insert by PCR.

Answer Id: E3317

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ceb20fd226a5000e4a18559916d50837_FAQ

Can N-terminal or C-terminal tags be attached to a Gateway® Entry clone?

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Answer

To have an N-terminal tag, the gene of interest must be in the correct reading frame when using non-TOPO® adapted Gateway® entry vectors. All TOPO® adapted Gateway® Entry vectors will automatically put the insert into the correct reading frame, and to add the N-terminal tag you simply recombine with a destination vector that has N-terminal tag.

To attach a C-terminal tag to your gene of interest, the insert must lack its stop codon, and be in the correct reading frame for compatibility with our C-terminal tagged destination vectors. Again, TOPO® adapted Gateway® Entry vectors will automatically put the insert into the correct reading frame. If you do not want the C-terminal tag to be expressed, simply include a stop codon at the end of the insert that is in frame with the initial ATG.

Generally, you need to choose a destination vector before you design and clone your insert into the Entry vector. This will determine whether you need to include an initiating ATG or stop codon with your insert.

Answer Id: E3950

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9fd12edd34b7e795629873843f6868a0_FAQ

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

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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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2333fd0dddc75b9ea6f234848a77567d_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

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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60186be7d45343b72b0be6a83da3bfad_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

Why does the pCR8/GW/TOPO® vector contain spectinomycin resistance? Do you offer spectinomycin?

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Answer

The pCR8/GW/TOPO® entry vector uses spectinomycin for selection so that the Entry clone that is generated can be used with any Destination vector. Most Destination vectors have ampicillin resistance although there are a few that have kanamycin or zeocin resistance. Otherwise, the background will be too high, unless the Entry vector is first linearized. Spectinomycin is a less commonly used selectable marker. We do not offer spectinomycin; spectinomycin dihydrochloride is available from Sigma (Cat. No. S4014).

Answer Id: E6813

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249887b914a4216657d554230955ab9b_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?

Product FAQ

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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Answer

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?

Product FAQ

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