12 votes
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What is the role of glucose in plasmid isolation?

This is a common misconception: in fact, in the alkaline lysis method for plasmid isolation, the glucose does not act as an osmotic stabiliser. Glucose is present in the resuspension buffer at 50 mM. ...
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11 votes
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What is the definition of a stringent/relaxed plasmid?

In short, yes, the definitions are still correct: The number of copies of a plasmid in the cell is determined by the mechanism of its replication: whether it is synchronized with the replication of ...
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10 votes

Does gene orientation relative to origin of replication matter on small plasmids?

I like this question, and I had a similar thought when reading that "Forward or Reverse Strand" post. I've since found a 2005 publication by Mirkin and Mirkin1, which investigates the ...
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  • 7,729
9 votes
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Is circular DNA the same as plasmids?

A plasmid is a length of circular or linear double stranded DNA that exists independent of chromosomal DNA with a cell (i.e. extra-chromosomal DNA) and often confers a selective advantage to an ...
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  • 106
8 votes
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Plasmid in the nucleus and gene expression

If you transfect cells with plasmid then these plasmids need to go into the nucleus (otherwise they wouldn't be transcribed). Getting the plasmid either happens during cell division (when the nucleus ...
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7 votes
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When do plasmids replicate relative to its host cell cycle?

I've found a nice review that has many details on plasmid replication in general, and several papers about pSC101 in detail, and I'll try to extract the key information from these papers. First of ...
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  • 3,078
6 votes
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Where can I find the pMON7124 plasmid sequence?

It seems that no good map of this plasmid is around. Life technology uses it in some of its bacterial strains, the quote: E. coli also contain the helper plasmid, pMON7124 (13.2 kb), which ...
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6 votes

Transformation of E.coli cells

The two plasmids have to have different origins of replication. Two plasmids from the same incompatibility group cannot survive together in the same cell. This quote is from the Wikipedia entry on ...
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  • 3,422
6 votes
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How does the DNA cross through bacterial cell wall during electroporation?

The bacterial cell wall is quite porous, and is not considered a permeability barrier for most small molecules. It mainly functions as structural support and to resist turgor pressures. The average ...
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  • 4,551
5 votes
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Transformation of E.coli cells

Double transformation (you may want to try searches using that term) has been doable for a very long time, but the efficiency will of course be less than either single transformation below the ...
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5 votes
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Can I use TBE to elute a plasmid shipped on filter paper?

In my experience it does not matter if you use water, TBE or TE buffer. Even with a little EDTA in it, it will not mess up enzymatic reactions as the concentration is way too low. TBE/TE buffer ...
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4 votes
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Low copy numbers of plasmids

I am sure you have done it; just a note to others: do a PCR screen to ascertain that the plasmid is there. For minipreps you generally do not need a starter culture. If you have screened your clones ...
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  • 35.1k
4 votes

What is the nature of plasmids?

You probably mean genomic and non-genomic DNA - Plasmids are small rings of non-genomic double-stranded DNA in Bacteria. They replicate mostly indepently of the genomic DNA can can occur from a few to ...
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4 votes
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What is the most critical step of plasmid purification?

There are a few critical steps (which sounds horrible if written together, but the method per se is robust and usually without problems): Resuspension of the pellet: Make sure it is really ...
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4 votes

How bad is ethidium bromide in your plasmid for downstream applications?

Purification and isolation of DNA bands by cutting them from agarose gel is commonplace (Lee et al., 2012). The purification step after excision of the band gets rid of most of the EtBr and other ...
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4 votes

Can bacteria pick up lethal plasmids?

The ability of bacteria to take up intact environmental DNA is called natural competence. One problem with trying to take advantage of this in a therapy is that it is not very efficient. Importantly, ...
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4 votes
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Can I pellet DNA back from dissolved state?

No, you cannot pellet dissolved DNA with ultracentrifugation. Yes, you can recover a pellet with additional treatments, similarly to how you got it in the first place; only instead of your input ...
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  • 5,690
4 votes
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How to replace intron region in a plasmid?

Your strategy would work, but if possible you might be better off using XmaI rather than SmaI, as the latter is a blunt-end cutting enzyme. Furthermore, your strategy would not replace the entire gpdA ...
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4 votes
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What volume of elution buffer for plasmid-containing filter paper?

The answer to this is... some. You only need a tiny amount of plasmid DNA to transform bacteria like DH5$\alpha$, typically in the 1-10 ng range, but much less, as low as about 10 pg (10 picograms) ...
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  • 7,773
4 votes

How do you read the gene insert domains on a plasmid from Addgene?

The GtCCR rhodopsin domain is shown by the thin brown and green arrows on the right of the image that overlap with the EYFP domain and are flanked by the CMV promoter and bGH poly(A) signal. These ...
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  • 7,773
4 votes

can mammalian expression vector be used in e coli to produce plasmid

Short answer: Looking at the plasmid map, yes, this should work. Long answer: It depends on the plasmid. To be able to replicate a plasmid in bacteria, you need a origin of replication (ori). To find ...
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4 votes

Plasmid Design and Integration events [Single vs Double cross over]

Single crossover is not the result of religation in the cell. The double-stranded break (DSB) in the plasmid instead stimulates a homologous recombination event, causing the entire plasmid to ...
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3 votes
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When should you use a stringent plasmid?

The three general advantages are: Lower expression. If your plasmid is expressing something toxic to cells at high levels, reducing the amount of DNA can reduce expression. Lower plasmid burden. If ...
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  • 4,470
3 votes

Plasmid choosing

PREFACE: There's a text, C. elegans: Methods and Applications, specifically doi: 10.1385/1-59745-151-7:109. They outline this exact experiment in it's essence, and one of the diagrams (page 110) notes ...
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  • 8,041
3 votes
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Identifying the origin of replication of an unannotated *E. coli* plasmid

Plasmapper Is quite good a recognizing common origins of replication. You will have to look up compatibility yourself. It is what I usually use when confronted with unnannotated plasmids.
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  • 1,405
3 votes

Is episome a plasmid or a virus?

I'll have to disagree with Alan. If the plasmid is integrated into a chromosome it doesn't lose its episomal status. Actually the integration of the plasmid is a characteristic of an episome. This is ...
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  • 161
3 votes

Is "TATA signal" synonymous with "TATA box"?

"Box" is an archaic term from long ago. It's little used. "Signal" has now replaced it. "TATA box" and "TATA signal" are synonymous.
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  • 141
3 votes

What, if any, is the difference between covalently closed circular DNA and plasmids?

As a descriptive term, we can take covalently closed, circular DNA (cccDNA) at face value: any DNA that is circular, with the ends joined by covalent bonds, can be described as cccDNA. Thus, plasmids ...
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  • 17.5k
3 votes
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Do all RNA polymerase in Eukaryotes share the same transcription factors?

Except for the TATA-binding protein (TBP), there are no common transcription factors for each RNA polymerase. However, there are homologies between many of the factors used by the different enzymes. ...
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  • 2,048
3 votes

Large plasmid ligation: do I need alkaline phosphatase?

A plasmid vector will always be prone to recircularisation when a single restriction enzyme has been used, but recombinant plasmids will also be formed during the ligation. Alkaline phosphatase ...
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