Questions tagged [plasmids]

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

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If the AraC protein was a gene repressor when it binds arabinose, would there be high or low transcription levels when arabinose is present?

If the AraC protein were to function as a gene repressor when it binds arabinose, would there be high or low transcription levels when arabinose is present?
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Is circular DNA the same as plasmids?

Chloroplasts have circular DNA, but would it be right to say that they have plasmids? Are plasmids and circular DNA even the same thing? Thank you in advance.
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How do you read the gene insert domains on a plasmid from Addgene?

This is a pretty basic question but I cannot find an answer anywhere. For plasmids on Addgene such as this one, there is supposed to be a GtCCR1 rhodopsin domain insert with a EYFP tag. However on the ...
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What volume of elution buffer for plasmid-containing filter paper?

I am attempting to elute a 9kb plasmid from a square of filter paper sent to me to be transformed into DH5a E. coli (photo below). Each filter paper square has been spotted with 1ug of plasmid DNA. I ...
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Does transcription terminate at the end of antibiotic resistance genes on the Duet expression plasmids?

I am working with the pETDuet and pRSFDuet plasmids to express RNAs under control of the T7 promoter. On these plasmids the antibiotic resistance genes (Amp for pET, and Kan for pRSF) are downstream ...
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T-vector creation

I'm working on TA cloning for the first time and I need to turn some existing plasmid backbones into T-vectors. I'm using a Klenow fragment (3' -> 5' exo-) to dA-tail my inserts. Is there any ...
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Can I use TBE to elute a plasmid shipped on filter paper?

I'm receiving a plasmid shipped to me dried on sterile filter paper, as described in this question. This protocol and many others like it call for eluting the DNA from the paper using TE buffer. My ...
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Identifying Promoters in Non-homologous Sequences within the Same Organism

In this paper, the authors cloned a GPD promoter for use in driving a GFP gene from Lentinula edodes using a set of provided primers. The primers were derived from the following accession GQ457137.1 ...
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How to replace intron region in a plasmid?

I'm considering working with the plasmid pRFHUE-eGFP and would like to replace the gpdA intron (which is the eGFP promoter region) with a promoter from another organism. What would be a good strategy ...
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Does direction relative to origin of replication matter on small plasmids?

The recent question about forward vs. reverse strand got me thinking about directionality conventions in synthetic biology. As noted in the answer to that question, if we consider only DNA in ...
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Calcium ions and bacterial transformation

What is the mechanism by which calcium ions and heat treatment allow the bacterial membrane to become permeable, allowing the uptake of plasmids?
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What's the difference (functionally speaking) between yeast centromeric plasmids and artificial chromosomes?

Historically, yeast artificial chromosomes (YACs) have been used for genome sequencing projects in the late 1990's. Nowadays, most researchers seem to use centromeric plasmids for expressing ...
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Is there any commercial vector technology platform for boosting recombinant protein production?

I am a novice in biomedical industry. Our team would like to search new vector technology for boosting recombinant protein production. It's difficult to google a firm that provide those plasmids. Dose ...
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PCR and large-scale PCR

I have performed PCR with 50 μL, which showed a clear band in the gel electrophoresis. I then performed large-scale PCR with 3650 μL, which showed an unclear band in the gel electrophoresis. What can ...
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How do we regulate the production of proteins when designing plasmids?

I think it should be no surprise that I, as many others, am interested in the new COVID-19 vaccines being developed. In my region of the world there are two mayor candidates. One is mRNA based and one ...
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Why we need a plasmid for r-DNA technology [closed]

Recently I was studying Biotechnology. When I went through the texts, I had a doubt: both plasmids and gene of interest are made of DNA stretches and bacteria directly absorb plasmids in a test tube (...
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What is the significance of running an uncut plasmid on electrophoresis gel?

What is the significance of running an uncut plasmid on electrophoresis gel? In this case we are talking about inserting a gene into plasmid, which then goes under PCR and then electrophoresis.
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Commercial suppliers of cloned vectors?

I am a recent transplant from academia to industry. I would like to follow-up on some experiments in the literature, for which I need cloned plasmids. Addgene of course doesn't (for the most part) ...
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What does Δcys mean after a gene name?

I am reading a paper and I have come across the following statement: "Plasmids encoding full-length NCAM140 and NCAM140Δcys, intracellular domain of NCAM140, and the NCAM140ID729–750 fragment ...
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Do most bacteria have plasmids?

Do most bacteria have plasmids? I googled that and I found this Yes, Plasmids naturally exist in all bacterial cells. Here But I know some of them don't! Is this sentence scientifically correct? ...
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How does the DNA cross through bacterial cell wall during electroporation?

There exists a lot of literature on electroporation of Gram-positive and negative bacteria. Most of it gives an explanation that electroporation works by creating transient pores in cell membranes of ...
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When purifying plasmid DNA after cloning, do you get endogenous plasmid in addition to your vector of interest?

Since bacteria naturally contain plasmids, when you do a cloning experiment and purify the plasmids at the end, do you get plasmid naturally present in that bacteria in addition to your vector? The ...
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Induce plasmid acquisition in bacteria to make them susceptible to antibiotics

Is it possible to use plasmids as a way of creating antibiotic susceptibility in bacteria. For example; in Microbiology, an introduction 13th edition by Pearson , Page 230 "the researchers traced the ...
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Why would a mammalian vector plasmid require an antibacterial resistance gene?

I have been trying to understand the composition of plasmids used in recombinant DNA cloning, such as this: https://www.genecopoeia.com/wp-content/uploads/2020/02/pReceiver-M35-051818.pdf and I am ...
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Why is Homologous Recombination (HR) more frequent at long sequence repeats?

I'm studying plasmids in bacteria (E. coli), and trying to understand the well-cited phenomenon that recombination frequency increases with longer repetitive sequences. I think this also applies to ...
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Can I pellet DNA back from dissolved state?

I have a dissolved plasmid pellet in water. Can I pellet it again by centrifuging it at 13000 rpm? If not, why?
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Which plasmid can express T7 RNA polymerase in mammalian cells?

Does anyone know what plasmid could express T7 RNA polymerase in mammalian cells, I would like to transfect it with other plasmid containing Gene Of Interest under control of T7 promoter. Thanks a lot!...
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Can bacteria pick up lethal plasmids?

I am sorry if this question is too general, and does not have any concrete answer. I was explaining to my non-biology-background friend about plasmids and how they are picked up by bacteria from the ...
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How can I improve efficiency of Ecoli transformation?

I am an intern in biology institute. I have a 17.3kbp plasmid need to transform to Ecoli. But I have tried many time but have no or very few colony on LB plate. I know large plasmid have less ...
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What is the origin of plasmids?

As I understand it, plasmids, like mitochondria, have their own genetic material and are capable of self-replication. According to Wikipedia: Plasmids are considered replicons, units of DNA capable ...
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Why don't mitochondria have plasmids?

According to the endosymbiotic theory, mitochondria are descended from specialised bacteria (probably purple nonsulfur bacteria) that somehow survived endocytosis by another species of prokaryote or ...
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Why pellet and resuspend E. coli for plasmid prep

For maxipreps, cant you just add all the stuff that would be in P1 (RNase A, EDTA) then just add P2? Because if we pellet cells then resuspend theres got to be a reason, right? Is it slats and other ...
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Join a linear plasmid just using a primer (without ligase enzyme)

Can I use just a primer and PCR to join the cutted plasmid like this ? without using any further enzyme like ligase. After the linear dsDNA plasmid annealed into 2 ssDNA, primer will bind to middle. ...
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use of antibiotics in selection of good plasmids

I have not understood the role of antibiotic medium in filtering the bacteria containing the recombinant plasmids with the ones having normal plasmids. Since all the plasmids of a certain bacterial ...
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History of plasmid maps

To create a small history of visual representations in biology, I am seeking the earliest published papers or books that have used plasmid maps as figures ; or strategies to effectively search for ...
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Can a plasmid cause cancer? [closed]

Can a plasmid cause cancer? According to wikipedia, plasmids are normally present in bacteria They (plasmids) are normally present in bacteria , and sometimes in eukaryotic organisms such as yeast [...
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579 views

When using a primer to replicate DNA in a plasmid, does it replicate the whole plasmid?

I've been learning about PCR and plasmids. I understand that the reason for having both a forward and a reverse primer is to extract and amplify the specific piece of DNA between these two primers. ...
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When doing minipreps, do "good" plasmids produce more DNA than "bad" plasmids?

I've been doing minipreps for close to 10 years using the standard qiagen kits and DH5a e. coli. I typically do 24 minipreps at a time and then identify the good plasmids by enzyme digestion, PCR, ...
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What is the role of potassium acetate in plasmid extraction?

What is the role of potassium acetate in the alkaline lysis method of plasmid extraction? Earlier I was thinking its role is to shield plasmid DNA (the same as sodium acetate in genomic DNA ...
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Dilute DNA plasmids with distilled water

I just extracted the DNA plasmid from E.Coli and dilute them in EB buffer. However, I've just figured out that the next step, which is electroporation, required that the DNA plasmid be diluted in ...
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Can you Interrupt a promoter region by inserting another promoter region into it?

I currently have a plasmid (pFPV25.1) which contains a promoter region with multiple cloning sights in it. I would like to place a new promoter I have isolated, and I was wondering whether I could ...
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Are there any alternatives to the Epicentre product Plasmid Safe?

I need to remove any traces of linear DNA (both single and double stranded) from a ligation reaction while keeping circular DNA intact. Up to now, I have used Epicentre's Plasmid Safe to do the job. I ...
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Are my plasmids single-stranded?

Hi, in this agarose gel photo (70 volts, 3.5uL Gel Red in 30ml of TBE, 1% Agarose, 1 hour run), I've checked the digestion of a large plasmid (15Kbp, one cut site). The theory says that the digested ...
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Large plasmid ligation: do I need alkaline phosphatase?

I have a doubt about a protocol I'm using for a ligation of a very large plasmid (15kbp circa) with an insert: after digestion with a single-cut enzyme (sticky ends), the protocol passes directly to ...
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What is the role of glucose in plasmid isolation?

My understanding is that glucose is used in the resuspension solution to prevent cells from bursting by maintaining the appropriate osmotic pressure. Why do we even bother doing this? The cells are ...
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Do all RNA polymerase in Eukaryotes share the same transcription factors?

I know that TFII proteins bind to cis elements in DNA and help initiate transcription for RNA polymerase II. But do RNA polymerase I and III also use these proteins when transcribing genes? Also, do ...
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Why Hfr cells are called high frequency recombination cells?

I understand that Hfr cells are formed when fragment of bacterial plasmid integrates itself in another bacteria's (host) genome through homologous recombination but my question is why do we call these ...
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LacZ' selection: blue colonies despite ligation of insert

It has been suggested that bacteria transformed with pBlueScript vector, containing an insert in the middle of the lacZ' gene, can still give blue colour on X-gal, if the insert is small and ligated ...
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Plasmid harbouring subunit S of type I R/M system

We have an assembly of a genome of a Lactobacillus species. The genome also contains a 7.5kb plasmid. Aside from the "usual plasmid genes" the plasmid contains: - toxin/antitoxin system - Subunit S ...
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Why do F- bacteria still exist?

When an F+ bacteria conjugates with an F-, it makes the other bactaria F+ too. So on the long run, all bactaria should be F+. Is there any mechanism that converts F- to F+? Could it be degeneration ...