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

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Answer

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: E3458

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efcaa31e653631377c2161f90ed6da61_FAQ

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

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Answer

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: E3459

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f06ad4e7b9e4557224e678ad3f01ca99_FAQ

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

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Answer

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: E3460

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c160a2cad726ee242d1488bf36ef8825_FAQ

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

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Answer

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: E3461

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bcab44ac634586605504795e8789bd25_FAQ

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

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Answer

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: E3476

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f6a7b3bf1a01aeaf3d25ba43ee8bbc60_FAQ

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

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Answer

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: E3474

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