I’m working with a lentiviral vector. What competent cells would you recommend using for propagation?

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We would recommend our Stbl3™ competent cells, as they have been tested for cloning of unstable lentiviral DNA sequences containing direct repeats.

Answer Id: E6705

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How can unstable or toxic DNA inserts be maintained in bacteria?

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There are a few steps you can take to improve stability of clones with difficult-to-maintain inserts. Supplement the medium with extra nutrients (e.g., add 20-30 mM glucose to Terrific Broth) or try a vector that has a reduced copy number (e.g., pBR322). Some clones can exhibit a high degree of deletions; this is usually a result of the clones having long terminal repeat (LTR) sequences or regions with high secondary structure. To overcome this problem, the cells can be grown at 30°C or ambient temperature (in LB or in a nutrient rich broth like Terrific Broth). Do not to let the cells reach late stationary phase in liquid culture. Alternatively, transform into cells that maintain unstable sequences such as Stbl2™, Stbl3™, or Stbl4™ cells.

Answer Id: E3099

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How much blasticidin do you usually put into culture medium to select for blasticidin-resistant clones for virus titration (HT1080 cells)?

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For HT1080 cells we typically use 10 μg/mL, but we strongly recommend that you generate a kill-curve for each antibiotic and cell line before proceeding. Most cell types respond to between 1 μg/mL and 10 μg/mL of blasticidin. For HT1080 cells, we typically use 100 μg/mL of Zeocin™ for Zeocin™-containing lentiviral vectors. But again, generation of a kill-curve is strongly suggested.

We strongly recommend titering on HT1080 cells to determine the absolute titer of infectious virus in your supernatant. The primary reason is that it's a way to standardize titers obtained in different labs. Transduction efficiency is high in these cells, and titering results are very accurate and reproducible, making HT1080 cells the gold standard for titering. You can then try different MOIs in other cell types based on HT1080 titers. For instance, you may require an MOI of 50 in one cell type or MOI of 10 in another cell type based on titers obtained in HT1080.Accurate titer, however, can be obtained in essentially any mammalian cell line, but 3T3 and HeLa cells have a lower transduction efficiency than HT1080 cells (for reasons unknown). Do not use 293FT cells for titering.

Answer Id: E4112

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Is S.O.C. medium absolutely required when recovering competent bacterial cells during transformation?

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Many media can be used to grow transformed cells, including standard LB, SOB or TB broths. However, S.O.C. is the optimal choice for recovery of the cells before plating. The nutrient-rich formula with added glucose is often important for obtaining maximum transformation efficiencies.

Answer Id: E3100

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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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What does the FT stand for in 293FT and why is this the most recommended producer cell line?

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The F stands for the high transfection efficiency of this particular 293 cell clone (called 293F) and the T stands for the SV40 large T antigen. If you want to use regular 293 cells or another 293T cell line, you will be able to produce virus, but the titers will be lower. The large T antigen expression plasmid is stably integrated in the 293FT cell and confers resistance to Geneticin™ antibiotic in these cells.

Answer Id: E4113

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What are the safety issues associated with the use of your viral systems?

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Both the lentiviral and adenoviral systems should be used following Biosafety Level 2 (BSL-2). We recommend strict adherence to all CDC guidelines for BSL-2 (as well as institutional guidelines). Life Technologies has also engineered specific safety features into the lentiviral system.

Consult the "Biosafety in Microbiological and Biomedical Laboratories" publication (www.cdc.gov, published by the CDC in the USA, describes BSL-2 handling) and the "Laboratory Biosafety Guidelines" publication (www.phac-aspc.gc.ca, published by the Centre for Emergency Preparedness and Response in Canada) for more information on safe handling of various organisms and the physical requirements for facilities that work with them.

Answer Id: E4099

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Why are 293FT cells cultured under Geneticin™ selection before transfection?

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For routine maintenance of 293FT cells, you need to add Geneticin™ (G418) antibiotic at a concentration of 500 μg/mL to maintain the Large T antigen plasmid/phenotype.

Answer Id: E4114

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What generation is your ViraPower™ lentiviral expression system? Can I use it with a 2nd generation lentiviral packaging mix?

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Our ViraPower™ lentiviral expression system is a 3rd generation system with regard to safety features. Our lentiviral expression vectors are derived from wild type HIV, but nearly all the wild type viral proteins (e.g., Vpr, Vpu, Vif, Nef, Tat) have been removed and the HIV envelope is not used. VSV-G (vesicular stomatitis virus G) envelope protein is used instead. Our ViraPower™ lentiviral expression system can be used with a 2nd generation lentiviral packaging mix. However, our lentiviral packaging mix would not be compatible with a 2nd generation lentiviral expression vector.

Answer Id: E6397

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I am getting small and large colonies after transformation of my lentiviral construct into Stbl3™ E.coli. Which colonies should I pick?

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Answer

We recommend picking the smaller colonies. Large colonies are usually the result of recombination events that result in loss of some part of the plasmid, conferring a growth advantage on the cells.

Answer Id: E6395

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How should I store lentivirus, adenovirus and viral vectors?

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Answer

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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Does the lentivirus produce any toxic viral genes?

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Answer

Lentiviruses produced with this system do not carry or express ANY viral genes and therefore have no associated toxicity issues. Only the protein expressed from the coding region between the LTR sites is incorporated into the mammalian cell chromosome and expressed. The lentivirus itself cannot replicate because of the built-in safety features.

Answer Id: E4116

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

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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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When should DMSO, formamide, glycerol and other cosolvents be used in PCR?

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Cosolvents may be used when there is a failure of amplification, either because the template contains stable hairpin-loops or the region of amplification is GC-rich. Keep in mind that all of these cosolvents have the effect of lowering enzyme activity, which will decrease amplification yield. For more information see P Landre et al (1995). The use of co-solvents to enhance amplification by the polymerase chain reaction. In: PCR Strategies, edited by MA Innis, DH Gelfand, JJ Sninsky. Academic Press, San Diego, CA, pp. 3-16.

Additionally, when amplifying very long PCR fragments (greater than 5 kb) the use of cosolvents is often recommended to help compensate for the increased melting temperature of these fragments.

Answer Id: E1320

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Why is it necessary to dilute ligated DNA products before adding them to competent bacterial cells?

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Answer

Components of the ligation reaction (enzymes, salts) can interfere with transformation, and may reduce the number of recombinant colonies or plaques. We recommend a five-fold dilution of the ligation mix, and adding not more than 1/10 of the diluted volume to the cells. For best results, the volume added should also not exceed 10% of the volume of the competent cells that you are using.

Answer Id: E3098

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