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V49220

Product FAQ

I’m getting few or no colonies, even with the transformation control. What could be the cause of this?

Answer

Ensure that the competent cells used were stored properly at -80 degrees C, and thawed on ice for immediate use. When adding DNA, mix competent cells gently: do not mix by pipetting up and down. Also do not exceed the maximum recommended amount of DNA for transformation (100 ng) or allow the volume of DNA added to exceed 10% of the volume of the competent cells, as these may inhibit the transformation.

Answer Id: E10027

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Product FAQ

I’m trying to anneal my oligos to create a ds oligo for ligation into one of your shRNA or miRNA RNAi vectors. When I run my ligated ds oligo on an agarose gel, I do not see any bands representing the ds oligo. What could be happening?

Answer

- Verify that the sequence of the bottom-strand oligo is complementary to the sequence of the top-strand oligo.
- For the shRNA vectors, make sure that you mix single-stranded oligos with complementary sequences. The top-strand oligo should include CACC on the 5’ end, while the bottom-strand oligo should include AAAA on the 5’ end.
- For the miRNA vectors, make sure that the top-strand oligo includes TGCT at the 5’ end and that the bottom-strand oligo includes CCTG at the 5’ end.

Answer Id: E10017

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Product FAQ

I am not getting any colonies after titering. What would suggest I try?

Answer

Perform a kill curve to determine the antibiotic sensitivity of your cell line. Ensure that viral stocks are stored properly at -80 degrees C, and do not undergo freeze/thaw more than 3 times. Lastly, transducer the lentiviral contruct into cells in the presence of Polybrene™ reagent.

Answer Id: E10028

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Product FAQ

I’m trying to create my entry clone but am seeing mutated inserts. What should I do?

Answer

We highly recommend sequencing positive transformants to confirm the sequence of the ds oligo insert. When screening transformants, we find that up to 20% of the clones may contain mutated inserts (generally 1 or 2 bp deletions within the ds oligo). The reason for this is not known, but may be due to triggering of repair mechanisms within E. coli as a result of the inverted repeat sequence within the ds oligo insert. Note: Entry clones containing mutated ds oligo inserts generally elicit a poor RNAi response in mammalian cells. Identify entry clones with the correct ds oligo sequence and use these clones for your RNAi analysis.
Mutated inserts could also be caused by using poor-quality single-stranded oligos. Use mass spectrometry to check for peaks of the wrong mass, or order HPLC- or PAGE-purified oligos to avoid this problem.

Answer Id: E10018

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Product FAQ

I’m seeing nonspecific, off-target gene knockdown. What should I do?

Answer

The target sequence used may contain strong homology to other genes; please select a different target region.

Answer Id: E10029

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Product FAQ

What are the safety features built into the BLOCK-iT™ Adenoviral RNAi Expression System?

Answer

The BLOCK-iT™ Adenoviral RNAi Expression System includes the following features designed to enhance its biosafety:

- The entire E1 region is deleted in the pAd/BLOCK-iT™-DEST expression vector. Expression of the E1 proteins is required for the expression of the other viral genes (e.g., late genes), and thus viral replication only occurs in cells that express E1 (Graham et al., 1977; Kozarsky and Wilson, 1993; Krougliak and Graham, 1995). This is where the Gateway™ Destination cassette is now located. The E3 region has also been deleted.
- Adenovirus produced from the pAd/BLOCK-iT™-DEST expression vector is replication-incompetent in any mammalian cells that do not express the E1a and E1b proteins (Graham et al., 1977; Kozarsky and Wilson, 1993; Krougliak and Graham, 1995).
- Adenovirus does not integrate into the host genome upon transduction. Because the virus is replication-incompetent, the presence of the viral genome is transient and will eventually be diluted out as cell division occurs.
- Despite the presence of the safety features discussed above, the adenovirus produced with this System can still pose some biohazardous risk since it can transduce primary human cells. For this reason, we highly recommend that you treat adenoviral stocks generated using this System as Biosafety Level 2 (BL-2) organisms and strictly follow all published guidelines for BL-2. Furthermore, exercise extra caution when creating adenovirus that express shRNA targeting human genes involved in controlling cell division (e.g., tumor suppressor genes) or when producing large-scale preparations of virus (See manual, pg. 11).
- For more information about the BL-2 guidelines and adenovirus handling, refer to the document, “Biosafety in Microbiological and Biomedical Laboratories”, 4th Edition, published by the Centers for Disease Control (CDC) (http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm)

Answer Id: E10013

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Product FAQ

Can I purchase the 5X LR Clonase™ buffer or 5X BP Clonase™ buffer separately?

Answer

We do not offer the 5X LR Clonase™ buffer and 5X BP Clonase™ buffer as standalone products. They are available as part of the enzyme kits.

Answer Id: E9855

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Product FAQ

I’m having difficulty sequencing the ds oligo insert in my shRNA construct. What is causing this, and do you have any suggestions on how to improve my sequencing results?

Answer

Difficulties sequencing could occur because the hairpin sequence is an inverted repeat that can form secondary structure during sequencing, resulting in a drop in the sequencing signal when entering the hairpin. If you encounter difficulties while sequencing, please try the following:

- Use high-quality, purified plasmid DNA for sequencing. We recommend preparing DNA using the Invitrogen™ PureLink™ HQ Mini Plasmid Purification Kit (Cat. No. K2100-01) or S.N.A.P.™ Plasmid DNA MidiPrep Kit (Cat. No. K1910-01).
- Add DMSO to the sequencing reaction to a final concentration of 5%.
- Increase the amount of template used in the reaction (up to twice the normal concentration).
- Standard sequencing kits typically use dITP in place of dGTP to reduce G:C compression. Other kits containing dGTP are available for sequencing G-rich and GT-rich templates. If you are using a standard commercial sequencing kit containing dITP, obtain a sequencing kit containing dGTP (e.g., dGTP BigDye™ Terminator v3.0 Ready Reaction Cycle Sequencing Kit, Cat. No. 4390229) and use a 7:1 molar ratio of dITP:dGTP in your sequencing reaction.

Answer Id: E10019

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Product FAQ

Are the BLOCK-iT™ miR RNAi Expression Kits compatible with adenoviral expression systems?

Answer

Yes. The miR miRNA vectors are Gateway™ cloning compatible, and you could use Gateway™ cloning to transfer the miR miRNA expression cassette to any of our Gateway™-adapted viral expression vectors.

Answer Id: E4646

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Product FAQ

I’m getting no fluorescence signal with my expression clone containing EmGFP. What should I do?

Answer

Please ensure that the recommended filter sets for detection of fluorescence are used. Use an inverted fluorescence microscope for analysis. If desired, allow the protein expression to continue for 1-3 days before assaying for fluorescence.

Answer Id: E10030

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Product FAQ

Will I get the same transduction efficiency with both lentivirus and adenovirus in the same cell line?

Answer

This depends entirely on the target cell. Adenovirus requires the coxsackie-adenovirus receptor (CAR) and an integrin for efficient transduction. Lentivirus (with VSV-G) binds to a lipid in the plasma membrane (present on all cell types). With two totally different mechanisms of entry into the cell, there will always be differences in transduction efficiencies. However, the efficiency of transduction for both viral systems is easily modulated by the multiplicity of infection (MOI) used.

Answer Id: E4102

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Product FAQ

How do I propagate and maintain the pAd/BLOCK-iT™-DEST vector?

Answer

We recommend using One Shot™ ccdB Survival T1R chemically competent cells (Cat. No. C751003) for transformation. This strain is resistant to ccdB effects and can support the propagation of plasmids containing the ccdB gene. To maintain integrity for the vector, select for transformants in media containing 50-100 μg/mL ampicillin and 15-30 μg/mL chloramphenicol.

Answer Id: E10014

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Product FAQ

How can I move my gene of interest from a Gateway™-adapted expression clone to a new Destination vector as I have lost the entry clone?

Answer

We would recommend performing a BP reaction with a Donor vector in order to obtain an entry clone. This entry clone can then be used in an LR reaction with the Destination vector to obtain the new expression clone.

Answer Id: E9856

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Product FAQ

Which competent E. coli do you recommend using for propagation of my Gateway™-adapted mammalian Destination vector?

Answer

We recommend using One Shot™ ccdB Survival™ 2 T1R Competent Cells, Cat. No. A10460. This strain is resistant to the toxic effects of the ccdB gene. Note: Do not use general E. coli cloning strains, including TOP10 or DH5alpha™, for propagation and maintenance, as these strains are sensitive to ccdB effects.

Answer Id: E9153

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Product FAQ

How do I know whether to choose lentivirus or adenovirus for viral expression?

Answer

If you're interested in stable integration and selection, choose the lentiviral system. We offer both a Directional TOPO™ (D-TOPO™) and Gateway™ version of the kit to provide flexibility in the cloning of the gene of interest.

If you're looking for transient gene expression, choose the adenoviral system. We offer the Gateway™ cloning method for this product. It should be noted, however, that gene expression from both systems is typically detected within 24-48 hours of transduction, so both systems can be used for experiments of a transient nature. The main difference is that lentivirus integrates into the host genome and adenovirus does not. Higher viral titers are achieved with the adenovirus.

Answer Id: E4098

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