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

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

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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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I’m able to see colonies on a plate, but when I pick them for liquid culture, no growth is observed. Why?

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One possible explanation could be toxicity associated with the insert. This toxicity does not affect slow growing cells on solid medium but is much stronger in faster growth conditions like liquid medium.

Suggestions:
1. Use TOP10F’ or any other strain with the LacIq repressor
2. Try using any other strain appropriate for cloning.
3. Lower growth temperature to 27 - 30 degrees C and grow the culture longer
4. Another possibility to explain lack of growth is possible phage contamination. In this situation we recommend using an E. coli strain that is T1 phage resistant like DH5alpha-T1R.

Answer Id: E6669

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

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

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

Answer Id: E4398

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Do I need to gel purify my PCR product for TOPO™ cloning?

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Gel purification is not required if the gel indicates that the PCR product is clean with no visible non-specific bands or primer dimers. It is recommended if the PCR product is >1.5 kb or if non-specific bands and primer dimers are visible on the gel. Smaller products clone much more efficiently into the vector than larger products; therefore, they should be eliminated from the sample prior to cloning. There is some reduction in A-overhangs if the PCR product is gel purified, which along with PCR product loss during the procedure may slightly reduce total number of colonies. However, the percentage of colonies with insert does not change; it is typically >90% recombinant clones.

Answer Id: E6745

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

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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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What are the advantages of using a TOPO™ TA cloning system compared to traditional TA cloning?

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Answer

TA cloning ligates the insert and vector using a T4 DNA ligase, while TOPO™ TA cloning uses the intrinsic properties of topoisomerase, which ligates the insert and vector during a 5 minute desktop reaction. TOPO™ TA cloning results in >95% recombinants, while TA cloning results in >80% recombinants.

Answer Id: E6742

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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?

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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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I see small satellite colonies on my LB+Amp plates. Why is this?

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Answer

These small colonies are most likely caused by degradation of the Ampicillin. The colonies are just untransformed cells that grow on LB with degraded Amp. In order to circumvent this scenario, you can try to:
1. Plate cells at a lower density
2. Use fresh LB-Amp plates or replace Ampicillin with carbenicillin.
3. The plates should not be incubated for more than 20 hours at 37 degrees C. Beta-lactamase, the enzyme produced from the Ampicillin-resistance gene, is secreted from the Amp-resistant transformants and inactivates the antibiotic in the area surrounding the transformant colony. This inactivation of the selection agent allows satellite colonies (which are not truly Amp-resistant) to grow. This is also true if carbenicillin is being used.

Answer Id: E6670

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

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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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I want to store my transformed cells long term. Do you have a protocol for this?

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Answer

For long-term storage, preparation of glycerol stocks stored at -70 degrees C is recommended. Follow the protocol below:
1. Pick one colony into 5 mL LB broth or S.O.C. medium. Grow overnight at 37 degrees C.
2. Prepare glycerol solution: 6 mL of S.O.B. medium and 4 mL of glycerol.
3. Take one volume of cells and add one volume of glycerol solution and mix.
4. Freeze in ethanol/dry ice. Store at -70 degrees C.

Answer Id: E6713

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

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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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How should I store my competent E. coli?

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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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