Often circular segments of DNA that can replicate outside of the chromosomes. Generally found in bacteria.

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Plasmid: Empty Backbone?

When searching for a plasmid on Addgene what does it mean if it is said to be an "empty backbone"? Why would one want an empty backbone?
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E. coli not growing in liquid medium

We are regularly doing bacterial transformation and subculture from plate to liquid media to extract DNA. This usually goes very well and is straightforward, but occasionally, the colonies that grew ...
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1answer
31 views

Identifying the origin of replication of an unknown *E. coli* plasmid

I have attempted a few searches for a list of origins of replication for plasmids in E. coli, but I was only able to find a list of origins, but not their individual sequences. The available plasmid ...
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1answer
19 views

Is it possible to express the cistrons from a polycistronic insertion fragment in a single plasmid?

I have a insertion fragment that I wish to express from pUC19 in Escherichia coli. The insertion fragment is a sub-section from a larger operon sequence and contains just the last two cistrons from ...
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48 views

When do plasmids replicate relative to its host cell cycle?

For plasmids is so much shorter than their host cell's genome (about 1/1000 in my case), it will take only 1/1000 time for it to replicate. With respect to cell cycle, when will that replication ...
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1answer
36 views

blue/white screening - results are opposite as expected

We inserted GFP-gene into plasmid Bluescript SK+, transformed E. coli with this construct, and then plated on an agar plate with Ampicillin and X-gal to do a blue/white screening. We got blue colonies ...
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39 views

Why are plasmid genes not already incorporated into bacterial chromosomes if necessary for stressful situations?

If plasmids are important for bacteria to express specific genes under stressful conditions, why are these genes not already incorporated into their chromosome to begin with? What is the evolutionary ...
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1answer
71 views

Plasmid in the nucleus and gene expression

If we insert a plasmid into a human nucleus that contains exact copy of gene and all relevant promoters to produce some human protein, will the cell create functional protein from that plasmid only ...
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65 views

Low copy numbers of plasmids

I have a plasmid with the P15A origin which apparently has a low copy number (see here). This would explain why my purification yeilds for subsequent digestion are low (gel shows the plasmid after ...
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65 views

What is the nature of plasmids? [closed]

What are the plasmids composed of? Are they composed of genomic or non-genomic DNA? I guess the answer is "genomic DNA" but I'm not sure.
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2answers
127 views

What are the rules for plasmid names?

What are best practices for naming newly created plasmids? For example, a common format is pABC123. What is the exact specification? Must there be 3 letters? What ...
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0answers
26 views

Gibson assembly - primer design with A and T rich regions

I have question about Gibson assembly. I have done it several times and it always worked okay for us, but now I want to assemble together a fragment which has sequence like this: ...
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113 views

What is the most critical step of plasmid purification?

During the process of plasmid purification and quantification, which step is the most critical to the success of the process?
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194 views

Where can I find the pMON7124 plasmid sequence?

I need the map of the pMON7124 plasmid. As this is not easy to find, I welcome any hints.
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223 views

The pBR322 convention

What is the pBR322 convention? For example in the description of the pET28a vector we can find information that the sequence is numbered by the pBR322 convention, so the T7 expression region is ...
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1k views

What is the definition of a stringent/relaxed plasmid?

I have found a publication which proposes some definitions, including a definition for strict and relaxed replication. The definitions are: Relaxed control of plasmid replication. Relaxed control ...
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3answers
115 views

Restriction endonucleases are found in?

Quoting from : Scientific American July 1975 The Manipulation of genes by Stanley Cohen : Restriction endonucleases (and modification methylases) are widespread in microorganisms; genes for ...
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1answer
4k views

What is a suicide plasmid?

What is a suicide plasmid? Example: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2737453/ mentions a "suicide vector". See also ...
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1answer
105 views

Is a sequential double transformation acceptable?

Standard protocol states having two compatible vectors being transformed simultaneously during the same procedure. I've come across a situation in which transforming one vector, obtaining results, and ...
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1answer
245 views

What is the minimum length required for a circular DNA ligation?

What is the minimum length of a DNA molecule for the ends to come in close enough contact that they can ligate. Assume there are free divalent cations in solution. Does anyone have any idea?
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1answer
84 views

Plasmid maintenance

I have obtained some plasmids used as integration vectors, this question may apply to all plasmids. I would like to have a somewhat continuos source for these plasmids, let's say that the origin of ...
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1answer
1k views

How does blue white screening work in selecting E coli that have taken up a plasmid vector?

The plasmid vector I am referring to is pCR 2.1 - TOPO. I added the vector to the E coli and plated them up onto LB+amp+X-gal plate, then incubated. After incubation the plates had two types of ...
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1answer
197 views

Expression of plasmid genes

Are plasmid genes always expressed? If so, then isn't a bacterium wasting it's resources in expressing genes (like antibiotic resistance) which are not required in "normal" conditions? If not, then ...
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1answer
74 views

How do plasmids protect themselves against restriction enzymes?

How do plasmids protect themselves from restriction enzymes released by bacteria (i.e., against bacteriophages)?
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2answers
97 views

Why do plasmids replicate on their own?

I know how plasmids can replicate independently of the main genome and know that they confer various properties to the bacteria and are useful in conjugation. My question is - what is the advantage ...
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2answers
334 views

What are common causes of unexpected ligation products?

I digested two plasmids, one with EcoRI and AgeI and the other with EcoRI and XmaI. Digests looked as expected, so I purified the respective fragments and set up the T4 DNA ligation (AgeI and XmaI ...
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1answer
199 views

Cloning two fluorescent proteins with different promoters in the same plasmid?

I want to clone two fluorescent proteins both driven by different promoters in a plasmid to be used to transform B. subtilis at the amyE locus. For this I want to use plasmid pSG1729 (Lewis and ...
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3answers
1k views

Primer design for Gibson assembly

I'm trying to design a primer for Gibson assembly. My gene of interest is on a plasmid, and I want to copy that gene, and put it into a different plasmid. I am unsure how to design my primers for ...
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2answers
3k views

Is episome a plasmid or a virus?

A plasmid is a small DNA molecule that is physically separate from, and can replicate independently of, chromosomal DNA within a cell. In general, in eukaryotes, episomes are closed circular DNA ...
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329 views

Do single crossovers occur in circular polynucleotides?

Single crossovers in circular pieces of DNA do not seem to be a big topic, because if they happened, they would lead to a kind of combined chromosome with two inner strands and one large outer strand. ...
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171 views

Vigorous shaking for HFR interrupted mating

I am trying to reproduce E. coli interrupted mating using an HFR strain, and I read that the cultures should be vigorously shaken at times to interrupt the DNA transfer from the F+(HFR) donors to the ...
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1answer
2k views

Co-transformation of plasmids from the same incompatibility group

Can two plasmids with the same origin of replication (for example pBR322 ori) and thus from the same incompatibility group be successfully co-transformed in E. coli? What are the mechanisms that would ...
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Restriction Mapping of Plasmid Assignment

pUWL22 is a circular, double stranded, 10.5 kb plasmid. It contains a gene encoding an enzyme that confers ampicillin resistance in the host bacterium. Cloning into the kpn I and Sst I sites ...
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3answers
12k views

How many agarose gel bands are typical for circularised DNA

I am aware that circular DNA can be both relaxed and super coiled. However when running an agarose gel of the circular plasmid along with singly digested plasmid with BamHI and HindIII, I see 1 band ...
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1answer
182 views

Taxonomy for plasmids

I liked the question about F' and R plasmids. What is a good resource that describes what is known about the functions and diversity of plasmids in the wild? Prokaryotes or eukaryotes? Wikipedia ...
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6answers
5k views

How should I ship plasmids?

I shipped 10 ┬ÁL of my vector miniprep to a collaborator in a 1.5 mL eppendorf parafilmed shut and stuffed into a 50 mL conical with some paper-towel padding. However, something happened on the way ...