Yes, you can use restriction enzymes Cla I (cutting at 1796) and BamH I (cutting at 2401) to remove the CMV promoter from the pLent6/V5-D-TOPO vector. Use Cla I and Spe I for the pLenti6/V5-DEST vector. Alternatively, we offer promoter-less lentiviral vectors that do not contain a promoter.

Identit de réponse : {0} E4111

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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.

Identit de réponse : {0} E4112

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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.

Identit de réponse : {0} E4113

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Coomassie dye does not bind well to phosphoproteins or other hydrophobic proteins. When using these proteins, switch to a silver stain to see total protein.

Identit de réponse : {0} E8361

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LMG194 will grow in LB and RM medium that contains M9 salts. LMG194 will not grow in M9 salts alone. RM medium is used to ensure low basal expression levels from the pBAD promoter.

Identit de réponse : {0} E3253

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TO stands for tetracycline operator, as this entry vector contains elements required for tetracycline-inducible expression of the shRNA in mammalian cells. The presence of the Tet operator sequences enables the shRNA of interest to be expressed in a tetracycline-dependent manner, thereby making this an inducible system.

Identit de réponse : {0} E9979

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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.

Identit de réponse : {0} E4114

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Below is a commonly used protocol:

(1) Add an equal volume of buffer-saturated phenol or phenol:chloroform:isoamyl alcohol (25:24:1, v/v/v) to an aqueous nucleic acid solution.

(2) Vortex, and centrifuge at 14,000 x g for 1 min to separate the phases.

(3) The concentration of NaCl in the aqueous solution should not exceed 0.5 M for good recovery of DNA.

(4) Residual phenol can be removed from the aqueous phase by extraction with an equal volume of chloroform or ether.

(5) After extraction, DNA is usually precipitated with ammonium acetate and ethanol.

Identit de réponse : {0} E4158

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The bottle contents must be mixed well before dispensing to ensure a uniform solution.

Identit de réponse : {0} E8362

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Any E. coli strain that is araBADC- and araEFGH+ is suitable for use with the pBAD promoter. Suitable strains that can be used include TOP10, TOP10F', and DH10B. Cells that are not araBADC- and therefore cannot be used with pBAD constructs, include DH5alpha, OmniMAX, Mach1, BL21(DE3) and INValphaF'.

Identit de réponse : {0} E3254

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You will need a double-stranded oligo that encodes the shRNA of interest to be cloned into one of the above-mentioned vectors. Use our RNAi Designer to design and synthesize two complementary single-stranded DNA oligonucleotides, with one encoding the shRNA of interest.

Identit de réponse : {0} E9980

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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.

Identit de réponse : {0} E4116

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For best results, DNA used in electroporation must have a very low ionic strength and a high resistance. A high-salt DNA sample may be purified by either ethanol precipitation or dialysis.

The following suggested protocols are for ligation reactions of 20ul. The volumes may be adjusted to suit the amount being prepared.

Purifying DNA by Precipitation: Add 5 to 10 ug of tRNA to a 20ul ligation reaction. Adjust the solution to 2.5 M in ammonium acetate using a 7.5 M ammonium acetate stock solution. Mix well. Add two volumes of 100 % ethanol. Centrifuge at 12,000 x g for 15 min at 4C. Remove the supernatant with a micropipet. Wash the pellet with 60ul of 70% ethanol. Centrifuge at 12,000 x g for 15 min at room temperature. Remove the supernatant with a micropipet. Air dry the pellet. Resuspend the DNA in 0.5X TE buffer [5 mM Tris-HCl, 0.5 mM EDTA (pH 7.5)] to a concentration of 10 ng/ul of DNA. Use 1 ul per transformation of 20 ul of cell suspension.

Purifying DNA by Microdialysis: Float a Millipore filter, type VS 0.025 um, on a pool of 0.5X TE buffer (or 10% glycerol) in a small plastic container. Place 20ul of the DNA solution as a drop on top of the filter. Incubate at room temperature for several hours. Withdraw the DNA drop from the filter and place it in a polypropylene microcentrifuge tube. Use 1ul of this DNA for each electrotransformation reaction.

Identit de réponse : {0} E4159

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Yes, any procedures that one ordinarily uses for coomassie-stained gels may be successfully performed on GelCode Blue Reagent-stained gels.

Identit de réponse : {0} E8363

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If the construct is growing under maximal repression (i.e. in LMG194 with D-glucose in RM media), the cells should be harvested and resuspended in RM medium containing 0.2% glycerol and the appropriate concentration of arabinose (determined empirically). If cells are growing in LB medium, then arabinose may be added directly to the medium. Note that TOP10 cells will not grow in RM medium.

Identit de réponse : {0} E3255

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