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

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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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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?

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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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How should I store lentivirus, adenovirus and viral vectors?

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Viral vectors:
Store lentiviral and adenoviral expression vectors at -20 degrees C. Due to their relatively large sizes, we do not recommend storing these vectors at -80 degrees C, as the vector solutions will completely freeze and too many freeze thaws from -80 degrees C will affect the cloning efficiency. At -20 degrees C, the vectors will be stable but will not freeze completely.

Virus:
Both adenovirus and lentivirus should be aliquoted immediately after production and stored at -80 degrees C.

Lentivirus is more sensitive to storage temperature and to freeze/thaw than adenovirus and should be handled with care. Adenovirus can typically be frozen/thawed up to 3 times without loss of titer, while lentivirus can lose up to 5% or more activity with each freeze/thaw. It is recommended to aliquot your virus into small working volumes immediately after production, freeze at -80 degrees C, and then thaw just one aliquot for titering. This way, every time you thaw a new aliquot it should be the same titer as your first tube.

Adenovirus can be kept overnight at 4 degrees C if necessary, but it is best to avoid this. Viruses will be most stable at -80 degrees C.

When stored properly, viral stocks should maintain consistent titer and be suitable for use for up to one year. After long-term storage, we recommend re-titering your viral stocks before use.

Answer Id: E4100

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

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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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Do you have a recommended single-step protocol for BP/LR recombination?

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Yes, we have come up with a single-step protocol for BP/LR Clonase™ reaction (http://www.thermofisher.com/us/en/home/life-science/cloning/gateway-cloning.html#1), where DNA fragments can be cloned into Destination vectors in a single step reaction, allowing you to save time and money.

Answer Id: E9857

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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?

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

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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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How does the adenoviral system work? How do I make an adenovirus expressing my gene of interest?

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Answer

Clone your gene of interest into the pAd/CMV/V5-DEST™ (or pAd-PL-DEST™ if you want to use your own promoter). Prior to cloning, if desired, propagate this vector in One Shot™ ccdB Survival™ 2 T1R Competent Cells (Cat. No. A10460) as described below. After cloning your gene of interest, propagate in E. coli strain TOP10. pAd/CMV/V5-GW/lacZ is provided as a positive control vector for expression.

Digest recombinant plasmid with Pac I to expose the ITRs (inverted terminal repeats).

Transfect (we recommend Lipofectamine™ 2000 reagent) E1-containing cells (293A cells) with linear DNA (only 10% of transfected cells will make virus).

Infected cells will ball up, and release virus to surrounding cells, which in turn will be killed and ball up. Look for plaques in the monolayer created by areas cleared by detaching, balled up cells (it takes 8-10 days to see visible plaques from this initial transfection).

Collect a crude viral lysate.

Amplify the adenovirus by infecting 293A producer cells with the crude viral lysate. Harvest virus after 2-3 days when cells ball up. Determine the titer of the adenoviral stock by performing a plaque assay. The virus generated is adenovirus type 5 (subclass C).

Add the viral supernatant to your mammalian cell line of interest to transduce cells.

Assay for recombinant protein of interest.

Once you have your gene of interest in the adenoviral vector, you can simply re-amplify when you need more of the virus. You do not need to repeat cloning steps and transfections each time.

When cloning or propagating DNA with unstable inserts (such as lentiviral DNA containing direct repeats), we recommend using the following modifications to reduce the chance of recombination between direct repeats:
- Select and culture transformants at 25-30 degrees C.
- Do not use "rich" bacterial media as they tend to give rise to a greater number of unwanted recombinants.
-If your plasmid confers chloramphenicol resistance, select and culture transformants using LB medium containing 15-30 μg/mL chloramphenicol in addition to the antibiotic appropriate for selection of your plasmid.

Answer Id: E4119

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Do I need to sequence my expression construct after transferring the U6 RNAi cassette into the pAd/BLOCK-iT™-DEST vector?

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Answer

This is not necessary, as the transfer preserves the orientation of the cassette. However, if you wish to sequence your DEST expression construct, we recommend the following primers:

Primer Sequence:
pAd forward priming site: 5’-GACTTTGACCGTTTACGTGGAGAC-3’
pAd reverse priming site: 5’-CCTTAAGCCACGCCCACACATTTC-3’

Answer Id: E10015

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Do you offer Gateway™ vectors for expression in plants?

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Answer

We do not offer any Gateway™ vectors for expression in plants.

Answer Id: E9854

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

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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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I’m seeing cytotoxic effects after transfection of my shRNA/miRNA construct. What is causing this?

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Answer

You can try to scale back the amount of transfection reagent used, or use a different reagent for the transfection. Additionally, ensure that the plasmid used is pure and properly prepared for transfection.

Answer Id: E10020

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Do you recommend a specific FBS for culture of the 293FT or 293A cells used in the ViraPower™ kits? What plastic plates do you recommend?

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Answer

We use mycoplasma-tested Gibco™ FBS (Cat. No. 16000-044) without any modifications. We have observed that when 293FT cells are cultured in the presence of this FBS following the instructions in the manual, virus production is better than that obtained with many other serum sources.

We use the following plasticware for 293A and 293FT cells:

T175--Fisher Cat. No. 10-126-13; this is a Falcon flask with 0.2 μm vented plug seal cap.

T75--Fisher Cat. No. 07-200-68; this is a Costar flask with 0.2 μm vented seal cap.

100 mm plate--Fisher Cat. No. 08-772E; this is a Falcon tissue culture-treated polystyrene plate

We get excellent adherence on these plates under routine cell culture/maintenance conditions (expect cell lysis in 293A cells when making adenovirus).

Answer Id: E4101

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Can I perform the single-step protocol for the BP/LR Clonase™ reaction using BP Clonase™ enzyme and LR Clonase™ enzyme instead of BP Clonase™ II enzyme and LR Clonase™ II enzyme?

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Answer

In the single-step protocol for the BP/LR Clonase™ reaction, we would not recommend substituting the BP Clonase™ II/LR Clonase™ II enzymes with BP Clonase™ /LR Clonase™ enzymes as this would result in very low recombination efficiency.

Answer Id: E9858

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

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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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