What enzymes could be used to cut the Neomycin resistance cassette out of the pcDNA™3.1 vector?

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Here’s a cloning scheme for cutting out the Neomycin resistance cassette from pcDNA™3.1 (-) vector, including the SV40 promoter and SV40 polyA signal:

At bp 1726 use either: Asp 700I, Mro XI, or XmnI (blunt). These enzymes also cut at 5107.
At bp 3237 use either: Bss NAI, Bst 1107I, or Bst 217I (blunt). Isolate the 1.5 kb fragment.

Answer Id: 3458

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e3ea33961a7c5b1ec04d6c97aa3b5379_FAQ

Why do I see expression of my protein in bacteria with pCDNA™3.1?

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It has been reported that pCDNA™3.1 may have a cryptic E. coli promoter that will often express your gene in E. coli when your gene is in the correct orientation for eukaryotic expression. The level of toxicity to E. coli will depend on your particular gene product and on the efficiency of translation initiation. This problem can be solved by placing a prokaryotic transcription terminator at the Hind III site, upstream of your gene. Eukaryotic expression will not be altered by this additional sequence.

Answer Id: 3459

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13d63838ef1fb6f34ca2dc6821c60e49_FAQ

What is the SV40 poly A recognition site for the Neomycin resistance gene in pcDNA™3.1 vector?

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pcDNA™3.1 vector, as with most vectors which contain what is described as the SV40 polyA region, actually contains 3 polyA signals, two on one strand and one on the other. Wild type SV40 virus has transcription going in both directions at different parts of its cycles (early and late), and transcripts from both directions get polyadenylated in the small region that contains these three signals.

In pcDNA™3.1 vector, the SV40 poly A recognition site for the Neomycin resistance gene is AAATAAA and is located at 3148 and 3177 in the SV40 Poly A.

Answer Id: 3460

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Which enzyme is recommended for the linearization of the pcDNA™3.1 vector in order to generate stable transfectants?

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Although not required, if you would like to linearize pcDNA™3.1 vector prior to stable transfection, Pvu I or Sca I are single-cutter enzymes that cut within the ampicillin resistance gene and should work well.

Answer Id: 3461

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Will the T7 promoter in mammalian expression vectors (for example, pcDNA3.1) function in prokaryotes for expression of proteins?

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No. While transcripts will usually be made, there is no ribosome binding site (RBS) or Shine-Dalgarno sequence to initiate translation. Furthermore, these vectors do not contain a prokaryotic transcription terminator.

Answer Id: 3476

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Can the Neomycin resistance gene found in your mammalian expression vectors also be used for kanamycin selection in E. coli?

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No, these vectors will not express kanamycin resistance in E. coli because the Neomycin resistance gene does not have a prokaryotic promoter. You cannot select transformants on kanamycin. This has been verified by streaking a glycerol stock of pcDNA™3.1 vector onto 3 plates: LB, LB + Amp, and LB + Kan. After incubation overnight at 37 degrees C, the cultures were observed to exhibit growth on LB and LB + Amp but no growth on LB + Kan.

Answer Id: 3474

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