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Are cDNA clones generated in pCMVSPORT6 (e.g. many of the IMAGE clones, non-Ultimate ORF full-length cDNA clones or clones generated by the SuperScript Plasmid system) fully Gateway-compatible? Product FAQ

Answer

These clones are not fully Gateway compatible. While the inserts are flanked by attB sites and can be transferred via BP reaction into Donor vectors, they are less than suitable for subsequent transfer into destination (DEST) vectors for protein expression, for the following reasons:

1. The inserts generally include a stop codon; hence C-terminal tagged DEST vectors are useless unless the intent is to express the insert in its native form (untagged).
2. The clones contain 5' and 3' untranslated regions of undetermined length, i.e. inserts were not specifically cloned into the Gateway reading frame. Even if the gene is in frame with a tag, the untranslated region would result in potentially several extra amino acids between the tag and the insert.
3. The 5' untranslated regions of the inserts may not contain a ribosome binding sequence (RBS) located 5-13 bases upstream of the ATG (a requirement for bacterial expression). While this does not apply to N-terminal tagged DEST vectors, which contain an RBS and ATG, it does apply to the use of C-terminal tagged vectors or vectors for native protein expression since they have no provision for an RBS upstream of the ATG.

In short, cDNA clones generated in pCMVSport6 can be transferred into mammalian DEST vectors for untagged native expression, or N-terminal tagged expression if the insert is found to already be in the Gateway reading frame (caveat - untranslated region will code for several amino acids). But for most other purposes, inserts should be amplified by PCR with primers directed at the ATG and stop codon regions and cloned into the appropriate pENTR/D-TOPO vector, or restriction-cloned in frame into the appropriate supercoiled pENTR vector.

Answer Id: E4897

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How does your Ultimate ORF collection differ from your other cDNA clones? Product FAQ

Answer

The cDNA clones we offer are Ultimate ORF clones, Full-Length human cDNA clones, MGC clones, and GeneStorm Expression-Tested clones. The Ultimate ORF clones, Full-length human cDNA clones, and the GeneStorm Expression-Ready clones are made in-house, whereas the MGC clones are derived from external sources and distributed by Thermo Fisher Scientific. The Ultimate ORF clones are fully-sequenced and guaranteed to match GenBank sequence information 100% at the amino acid level, whereas the sequence of the other cDNA clones we offer is not guaranteed.

Answer Id: E6776

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Which clone collection should I pick: Ultimate ORF, Full-length human cDNA, or MGC? Product FAQ

Answer

We recommend choosing the Ultimate ORF clones if quality and sequence are your main priority. Further, these clones are provided in a Gateway vector backbone, so it is easy to shuttle the insert into multiple host systems. The Full-length human cDNA clones and MGC clones are only 5´ and 3´ end-sequenced, but Full-length human cDNA clones offer the advantage of being in a Gateway Entry vector backbone. MGC clones are available for human, mouse, and rat species; Ultimate ORF clones are available for human and mouse species and Full-Length human cDNA clones are available only for human species.

Answer Id: E6772

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How do I find/order an individual BAC/PAC clone? Product FAQ

Answer

There are several internet sites that may be used to locate BAC/PAC clones. One of them is listed below:

- Go here.
- Select the species of interest from the “Select organism” box.
- Type the feature, location or gene name in the “Search in genome” box on the right.
- As an example, choose “Search in genome”, enter “ITGA5” as the “Feature name” and click the search icon.
- Click on “Tracks shown”.
- Find the Clone library of interest, click on “Genomic Clones” and then select the box for the library of interest: for example: RP23 (RPCI-23 Female (C57BL/6J) Mouse BAC Library) and click “Configure”.
- In the refreshed window, you will see a list of BAC/PAC clones containing the ITGA5 gene (for example: RP23-427P19 where the Clone ID is 427P19).
- Go to CloneRanger, select the “BAC Mouse RP23” Collection, type in the Clone ID (in this case, 427P19), and click “Submit” to see if we carry the BAC clone. If the order is being placed by phone, provide the Cat No. (RPCI23.C) and the Clone address (in this case, RP23-427P19).

Answer Id: E16847

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My MGC clone is in the NCBI database, but I cannot find it using the CloneRanger tool. What could be the possible reason? Product FAQ

Answer

– We may have had this clone in the past, but it might have been removed from the collection because it was a “no grow”
– If it is a recently created clone, it might take us a while to stock it. We only offer clones that have been entered into the NCBI database more than 6 months to 1 year ago.
– Please check with ATTC (www.attc.org) or Open Biosystems (www.openbiosystems.com) to see if they carry the clone.

Answer Id: E6781

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What is the name of the backbone vector that is used for your Ultimate ORF clones? What are its main features and benefits? Product FAQ

Answer

The backbone vector that is used is pENTR221, which is a Gateway Entry vector. The insert (an open reading frame that begins with ATG and ends with a TAG stop codon) is flanked by attL1 and attL2 sites. The construct is ready for Gateway LR recombination with a DEST vector containing attR1 and attR2 sites. It is compatible with N-terminal tagged DEST vectors but not with C-terminal tagged DEST vectors. To express the C-terminal tag, the Tag-On-Demand technology would have to be used or the TAG stop codon would have to be mutated. In place of the Tag-On-Demand adenovirus product we sold previously, we offer the vector expressing the tRNA suppressor as a custom service. If you are interested in this service, please e-mail custom.services@lifetech.com.

Answer Id: E6777

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How can I find more information about the history of the library from which my MGC clone was derived? Product FAQ

Answer

Please go to the NIH Mammalian Gene Collection Consortium website at http://mgc.nci.nih.gov.

Answer Id: E6775

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What are some reasons why I should pick a genomic BAC clone for my gene instead of a cDNA clone? Product FAQ

Answer

BAC clones are genomic clones containing very large inserts (>100 kb) that may contain several genes, whereas cDNA clones only contain a single open reading frame of a gene. BAC clones are used for promoter and intron/exon analysis, chromosome localization, and making transgenic mice.

Answer Id: E6778

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Do you offer pcDNA 3.3-TOPO TA Cloning Kit (Cat. No. K830001)? Product FAQ

Answer

pcDNA 3.3-TOPO TA Cloning Kit (Cat. No. K830001) has been discontinued. As an alternative, you may use pcDNA 3.4 TOPO TA Cloning Kit (Cat. No. A14697).

Answer Id: E19274

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What is the difference between pcDNA 3.4-TOPO TA and pcDNA 3.3-TOPO TA vectors? Product FAQ

Answer

pcDNA 3.4-TOPO TA vector is an improvement over pcDNA 3.3-TOPO TA vector. It contains the WPRE (Woodchuck Posttranscriptional Regulatory Element) that allows for 2- to 3-fold higher levels of expression than pcDNA 3.3-TOPO TA vector.

Answer Id: E9225

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Are there suggestions to increase the efficiency of cloning a long PCR insert into a TOPO vector (other than using TOPO XL-2 PCR cloning kit)? Product FAQ

Answer

1. Large inserts should be gel-purified prior to cloning. Gel purification of long PCR products often improves efficiency, as smaller non-specific bands that may not be visible on the gel will interfere with cloning of the insert of interest.

2. Cloning efficiency of gel-purified products can be further increased by avoiding visualization of the bands with UV light. Ethidium bromide and UV light can cause nicking of the gel-isolated DNA, and nicked DNA is a poor substrate for cloning - it is degraded much more efficiently by nucleases in the bacteria.

One option is to purify from a gel stained with SYBR Safe stain and visualized with blue light on the Safe Imager. Data showing improved cloning efficiency after gel purification with this method versus ethidium bromide and UV light is available on the Thermo Fisher Scientific website at www.thermofisher.com/safeimager.<

Another option is to stain the DNA with crystal violet - the pCR-XL TOPO Cloning kits and SNAP UV-Free Gel Purification Kit (Cat. No. K2000-25) use crystal violet instead of ethidium bromide and UV to visualize the PCR product during gel isolation. Crystal violet is also available separately from Sigma (Cat. No. C3886), and can be used for gel purification of PCR products for cloning into any TOPO-adapted vector.

If neither of these options is convenient, try to use a minimal amount of EtBr in the gel, only enough to visualize the fragment of interest. 0.5 µL of a 10 mg/mL stock of EtBr per 100 mL of agarose gel is generally sufficient.

3. Keep the insert:vector molar ratio low, optimally 1:1, a maximum of 3:1 to 5:1 ratio. If the concentration of insert is too high, one end of the PCR product is able to bind to one end of the vector, and the other end of the PCR product would then have to compete with the other free-floating PCR products in the solution for the other end of the vector. This competition becomes more significant as the size of the insert increases.

4. Increase incubation time of the TOPO reaction to either 1 hour, 2 hours, or even overnight incubations at room temperature. These longer incubations have been used without any detrimental effects as long as the Salt solution is used in the TOPO reaction. Longer incubations generally increase the cloning efficiency for longer PCR fragments; shorter fragments show little benefit from these longer incubations.

5. Dilute the reaction to 20 µL, while maintaining the same amount of vector and insert. Increase salt solution volume to 3.7 µL to compensate for the increase in volume, and add sterile distilled water up to 20 µl. Diluting the reaction reduces the competition for the vector ends further.

Any one or combination of the above strategies should improve the efficiencies of cloning larger inserts in a TOPO reaction. Keep in mind that the TOPO reaction does have a strong size bias, and that any smaller fragments present in the PCR reaction would clone much more efficiently than the fragment of interest, even if the smaller fragment is not readily visible on a gel. Therefore, it is highly recommended that the PCR product of interest be gel-purified.

Answer Id: E3937

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How are GeneArt synthetic libraries supplied? Product FAQ

Answer

Two standard options are available:
- As a set of linear DNA fragments, ready for restriction enzyme digestion and subcloning.
- Subcloned into any vector and delivered as a glycerol stock and plasmid preparation.

Answer Id: E11570

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Can the GeneArt Seamless Cloning system be used for library construction? What about for cloning of random DNA libraries made from oligos? Product FAQ

Answer

In theory, the GeneArt Seamless Cloning system can be used for library construction but we have not tested either application. Adapters with the required homology to the cloning vector would have to be generated.

Answer Id: E6825

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What is the difference between the GeneArt Seamless PLUS Cloning and Assembly Kit and the original GeneArt Seamless Cloning and Assembly Kit? Product FAQ

Answer

The GeneArt Seamless PLUS kit is an improved version of the original kit. It is recommended for the assembly of up to 4 fragments and a vector totaling up to 40 kb compared to 13 kb for the original. The GeneArt enzyme mix is also provided as a 2X mix with buffer that can be stored at -20 degrees C instead of -80 degrees C. Finally, the kit comes with the linearized pYES7L vector and Stbl3/pRK2013 cells that allows for horizontal transfer of the construct into a variety of recipient strains. To select the GeneArt Seamless Cloning and Assembly kit that is best for your application, please visit https://www.thermofisher.com/us/en/home/life-science/cloning/seamless-cloning-and-genetic-assembly.html.

Answer Id: E6822

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Is the cloning efficiency with the GeneArt Seamless Cloning and Assembly kits different between shorter and longer fragments? Product FAQ

Answer

We suspect that there would be some degree of preference for shorter fragments. We have seen 100% cloning efficiency with a 5 kb fragment, but the colony output was lower when compared to a 2 kb fragment. For example, you get about 400 colonies per 1 µL reaction for 5 kb and about 1200-2000 colonies per 1 µL for 2 kb. Also, we have observed in assemblies of larger fragments like 5 kb that if the PCR reaction of the 5 kb fragment is not gel-purified and there is a significant PCR band at a smaller size, then the smaller fragment tends to go in more than the 5 kb. We have not observed anything like this in fragments of 1 kb or 2 kb.

Answer Id: E6826

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