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

Is it okay if my construct has an ATG that is upstream of the ATG in my gene of interest? Will it interfere with translation of my gene?

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Answer

Translation initiation will occur at the first ATG encountered by the ribosome, although in the absence of a Kozak sequence, initiation will be relatively weak. Any insert downstream would express a fusion protein if it is in frame with this initial ATG, but levels of expressed protein are predicted to be low if there is a non-Kozak consensus sequence. If the vector contains a non-Kozak consensus ATG, we recommend that you clone your gene upstream of that ATG and include a Kozak sequence for optimal expression.

Answer Id: E9180

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16c54268c5857e0549e9da663fb0c2ef_FAQ

I am using a mammalian expression vector that has the neomycin resistance gene. Can I use neomycin for stable selection in mammalian cells?

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Answer

No; neomycin is toxic to mammalian cells. We recommend using Geneticin® (a.k.a. G418 Sulfate), as it is a less toxic and very effective alternative for selection in mammalian cells.

Answer Id: E9181

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86410c89d41682a89d98002342b5c3f1_FAQ

I used a mammalian expression vector but do not get any expression of my protein. Can you help me troubleshoot?

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Answer

Here are possible causes and solutions:

- Try the control expression that is included in the kit
Possible detection problem:

- Detection of expressed protein may not be possible in a transient transfection, since the transfection efficiency may be too low for detection by methods that assess the entire transfected population. We recommend optimizing the transfection efficiency, doing stable selection, or using methods that permit examination of individual cells. You can also increase the level of expression by changing the promoter or cell type.
- Expression within the cell may be too low for the chosen detection method. We recommend optimizing the detection protocol or finding more sensitive methods. If the protein is being detected by Coomassie/silver staining, we recommend doing a western blot for increased sensitivity. The presence of endogenous proteins in the lysate may obscure the protein of interest in a Coomassie/silver stain. If available, we recommend using a positive control for the western blot. Protein might be degraded or truncated: Check on a Northern. Possible time-course issue: Since the expression of a protein over time will depend upon the nature of the protein, we always recommend doing a time course for expression. A pilot time-course assay will help to determine the optimal window for expression. Possible cloning issues: Verify clones by restriction digestion and/or sequencing.

Answer Id: E9182

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78bbc5bb321b11c2f6a40a2e143aae2a_FAQ

I performed stable selection but my antibiotic-resistant clones do not express my gene of interest. What could have gone wrong?

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Answer

Here are possible causes and solutions:

Detection method may not be appropriate or sensitive enough:
- We recommend optimizing the detection protocol or finding more sensitive methods. If the protein is being detected by Coomassie/silver staining, we recommend doing a western blot for increased sensitivity. The presence of endogenous proteins in the lysate may obscure the protein of interest in a Coomassie/silver stain. If available, we recommend using a positive control for the western blot.
- Insufficient number of clones screened: Screen at least 20 clones.
- Inappropriate antibiotic concentration used for stable selection: Make sure the antibiotic kill curve was performed correctly. Since the potency of a given antibiotic depends upon cell type, serum, medium, and culture technique, the dose must be determined each time a stable selection is performed. Even the stable cell lines we offer may be more or less sensitive to the dose we recommend if the medium or serum is significantly different.
- Expression of gene product (even low level) may not be compatible with growth of the cell line: Use an inducible expression system.
- Negative clones may result from preferential linearization at a vector site critical for expression of the gene of interest: Linearize the vector at a site that is not critical for expression, such as within the bacterial resistance marker.

Answer Id: E9183

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559c6028788610ad1cbc4bc184da429d_FAQ

I am working with a mouse cell line and would like to express my gene at high levels using one of your vectors with the CMV promoter. Do you foresee any problems with this approach?

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Answer

The CMV promoter is known to be downregulated over time in mouse cell lines. Hence, we recommend using one of our non-CMV vectors, such as those with the EF1alpha or UbC promoter, for long-term expression in mouse cell lines.

Answer Id: E9152

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aa6000854a733631ad122a7c944854d0_FAQ

Do you offer a GFP-expressing mammalian expression vector that I can use as a control to monitor my transfection and expression?

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Answer

We offer pJTI™ R4 Exp CMV EmGFP pA Vector, Cat. No. A14146, which you can use to monitor your transfection and expression.

Answer Id: E9154

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2c85f53110326245a2efcdaf9c503be0_FAQ

What is the difference between pcDNA™3 and pcDNA™3.1 vectors?

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Answer

pcDNA™3 is no longer available from Thermo Fisher Scientific but has been directly replaced by pcDNA™3.1, which was derived from pcDNA™3. The center of the multiple cloning site (MCS) within the original pcDNA™3 vector contained homology to a hairpin mRNA structure and involved the Eag I, Not I, and both BstXI sequences. This hairpin would only have affected expression of genes cloned downstream of the Not I site, if at all. To address this issue, some sequences were removed, including the Eag I site, and the BstXI sequences were slightly modified to reduce homology. A 32-base fragment from pcDNA™3 (between bases 995 and 1026), which contains the Sp6 primer site, was also removed and 11 bases were inserted in its place, adding another PmeI restriction site into the MCS of pcDNA™3.1.

Answer Id: E9155

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57c117aa0660a6a5889c758141d95ac4_FAQ

What is the significance of the (+) and (-) designations for the pcDNA™3.1, pcDNA™3.1/Zeo, and pcDNA™3.1/Hygro vectors?

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Answer

The (+) and (-) designations refer to the orientation of the multiple cloning sites in these vectors. The availability of the cloning site in two orientations facilitates flexibility in cloning scheme design, so that if, for example, your insert must clone in as a Not I to Bam HI orientation, you may choose the (-) version of these vectors.

Answer Id: E9156

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d766ca5f7254252fc6fb7a894095747a_FAQ

What is the difference between pcDNA™3.1 vectors and the pcDNA™3.3-TOPO® vector?

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Answer

pcDNA™3.1 vectors contain the core CMV promoter that is truncated before the start of transcription, whereas the pcDNA™ 3.3-TOPO® vector has the 672 bp native CMV promoter. This native CMV promoter allows high-level gene expression with two- to five-fold higher protein yields compared to other expression vectors. pcDNA™3.1 vectors are available in restriction, TOPO®, and Gateway® cloning versions and as untagged and epitope-tagged versions, whereas the pcDNA™3.3-TOPO® vector is a TOPO® TA-adapted, untagged vector that can be used to express native proteins without extraneous amino acids, and is hence ideal for antibody production and structural biology.

Answer Id: E9157

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f7ae83c62f53fe33370165ecf400ff07_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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1fc4b5923440e42f585666cdee1d9071_FAQ