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

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Full Sequence for Plasmid pMG101

I am trying to find the full plasmid sequence for pMG101. I have been looking through other papers that have sequenced this plasmid. The GenBank access numbers I got are the following: AY009372–...
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25 views

How to specify a promoter in de novo gene synthesis service?

I am trying to use a de novo gene synthesis service (Genscript) and one part confuses me: Where do I pick the choice of promoter? Or is the promoter choice automatic based on my plasmid choice? e.g....
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13 views

Restriction enzyme recognition sites for plasmid libraries

I'm having trouble with the following question: You wish to cleave genomic DNA into fragments average about 2kb for cloning into a plasmid library. Which of the following restriction enzyme ...
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37 views

Can we modify plasmids to eliminate antibiotic resistance? [closed]

This is a very general question regarding the potential use of artificially modified plasmids to block or modify bacterial evolution in order to prevent development of anti-biotic resistance. For ...
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25 views

Complementing back the expression of a knockout gene

I have a human cell line (Huh7.5, hepatoma-derived) in which Ago2 gene has been knocked out by the Crispr/Cas9 technology. I checked the effective gene editing by sequencing and I also confirmed the ...
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64 views

Can Bacteria Repair Dephosphorylated Plasmids?

I have been trying to insert a short sequence of DNA into a plasmid. I wanted to make two different versions, with the insert at different locations. I chose HinDIII and SalI sites approximately ...
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20 views

When should you use a stringent plasmid

I was wondering if anyone had good examples of when you would want to use a stringent plasmid vs a relaxed plasmid in a research setting. Thanks
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283 views

What is the difference between a bacterial artificial chromosome (BAC) and a plasmid?

Is it just that a BAC is generally larger and artificially constructed? Or are there any other differences?
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99 views

Enzymes and plasmids

A high school class has analyzed plasmids by the help of restriction enzymes and electrophoresis. The class got delivered two different plasmids, pBR 322 and pC 508. These two plasmids were going to ...
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2answers
78 views

Plasmid addiction system in high-copy number plasmids

Are there high-copy number plasmids that utilize a plasmid-addiction system?
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46 views

What is the sequence of pPROLar.A plasmid?

I've been looking for the sequence of pPROLar.A122 plasmid but I haven't found it. Where could I find the sequence? The first link (addgene) on googling reports a plasmid which is reported to have ...
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38 views

Plasmid Expression Vector [closed]

If I am going to transform a plasmid into a bacterium, which will then be fed to a C. elegans, the expression vector needs to be bacterial correct? The other option is to have a worm expression vector,...
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41 views

Question about cutting gene in Plasmid

I am designing a researching proposal for the class. Because it uses microinjection to up-regulate the gene in C. elegans, the plasmid pCFJ104 - Pmyo-3::mCherry::unc-54 sequences has been chosen. But ...
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79 views

Plasmid choosing

To design a experiment in feeding of C. elegans. It has to choose a plasmid vector to insert the gene of interest that can feed to C. elegans. Many paper are using pL4440 for the feeding vector, ...
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2k 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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0answers
55 views

Why is there a size limit on inserts that a plasmid can accept?

Throughout my undergraduate education I have been taught that plasmids can't carry very large inserts, but I have never been told why. Any insight would be greatly appreciated.
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37 views

Plasmid with ITPG and pBR322 ori

I'm looking for suggestions as my internet searches aren't turning up much. I'm trying to find a plasmid that has the pBR322 origin, as it's low(ish) copy number and can tolerate large inserts. ...
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72 views

Plasmid copy number and Rop protein

If i want to transform a bacteria (E. coli) with a particular plasmid (in my case pBR322) will the presence of the Rop gene affect the production of it ? Is it desirable to use a plasmid without that ...
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307 views

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

I want to transfect Adipose Derived stem cells using a clone of my own. My problem is that I get a lot of contamination (not only genomic, but due to the nature of the technique, I get undesired ...
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4answers
665 views

Identifying the origin of replication of an unannotated *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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2answers
429 views

Transformation of E.coli cells

As a team of undergraduate researchers we are looking to transform two genes of interest into our competent E.coli cells. Both of these genes are in separate plasmids and we would like to put them ...
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393 views

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
467 views

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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2answers
5k 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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57 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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215 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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241 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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79 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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1answer
107 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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277 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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303 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
5k 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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138 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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2answers
541 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
31 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: 5'...
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335 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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393 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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521 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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3answers
134 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
9k 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 http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/plasmids/allele-...
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2answers
119 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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268 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
540 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
118 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
265 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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2answers
610 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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90 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
2k 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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3answers
15k 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
467 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. ...