Tagged Questions

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

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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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1answer
30 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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1answer
95 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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1answer
87 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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1answer
259 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
105 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
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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
64 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 ...
4
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1answer
171 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
78 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
713 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
139 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
65 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
81 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
180 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
156 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 ...
2
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3answers
950 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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1answer
2k 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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1answer
267 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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1answer
150 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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2answers
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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
9k 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 ...
8
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1answer
161 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
4k 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 ...