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Questions tagged [plasmids]

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

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Production method of custom seed mir451? How to edit middle of a fragment?

I am trying to create a plasmid that produces mir451 targeting a new gene, so I need to alter the seed sequence. The seed sequence is 7 bp long, so I think that is too long for PCR mutagenesis. How do ...
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21 views

Which is the best results for Plasmid dna extraction on gel electrophoresis?

I did plasmid dna extraction and uploaded on the gel. Well 1: marker Well 2: nicked,circular,and supercoiled bands Well 3: linear,circular,and supercoiled bands Well 4: circular and supercoiled bands. ...
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1 answer
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What is a good expression vector for Salmonella enterica subsp. enterica serovar typhimurium

I am interested in expressing custom proteins in a Salmonella strain, however I am facing difficulties in finding the appropriate expression vector for it. It seems that most of the resources provide ...
2 votes
1 answer
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How to separate two plasmids from E coli with the same backbone?

I'm using transformation-associated recombination (TAR) in yeast to capture a biosynthetic gene cluster (32 kb) by transforming gDNA and a capture vector (11 kb) with homology arms. I identified a ...
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Problems reading my gel electrophoresis result

This is an image of my gel electrophoresis result. I used the alkaline lysis method to extract E. coli plasmid and put it on gel electrophoresis. Based on what I had learned, I expected to see a band ...
3 votes
1 answer
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Use of plasmid pXen5 for transposon screening

I would like to use the plasmid pXen5 (by Xenogen) for a transposon screen. It contains two inverted repeat sequences, with Luciferase, Kanamycin, and the transposase itself in between. (It's tn1409). ...
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1 answer
51 views

What are possible tweaks when a protein expression from a plasmid shipped on paper fails?

In my group we recently tried to express a protein necessary to perform an analytical method in BL21(DE3). The first two attempts did fail and no colonies did grow on the agarose. I hear, that the ...
2 votes
1 answer
88 views

Can introns be removed when designing protein expressing episomal plasmids?

If your goal is to cause a specific protein to be expressed transiently via an episomally maintained gene, could you just exclude all introns and the exons not associated with that transcript variant ...
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What is the enzyme production rate of a single E. Coli bacterium?

Suppose an E.Coli bacterium intakes a plasmid which encodes for an enzyme, if the promoter strength is known, is is possible to predict the rate at which the enzyme is produced? Is there an equation ...
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367 views

Migration of cut vs. non-cut plasmid on gel

As you might know there are 3 common forms of a plasmid: ccc-form (CCC), being supercoiled. oc-form (OC), being nicked and therefore relaxed. linearized form (L), being cut on both strands and linear....
2 votes
1 answer
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Linearising plasmids for CRISPR experiment

I am currently designing a mock CRISPR knock-out experiment, and I’m wanting to insert a plasmid for selection. Using a restriction enzyme at 2750bp for cutting, would the location of the cut site ...
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Can the genetic sequences for CRISPR components be inserted into the host genome so that the cell perpetually produces CRISPR components?

Theoretically speaking, can you insert the gene sequences for cas9, sgRNA, and promoters into the host genome so that the cell perpetually produces CRISPR components? In this scenario, I'm guessing ...
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1 answer
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Plasmids for high school students?

For a final project in AP Biology, I am planning to design an experiment that uses the plasmid #170380 from Addgene. However, under availability they say "Academic Institutions and Nonprofits ...
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1 answer
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Cut plasmids with unknown sequence only once

Is there a way to linearize plasmids in a sequence-independent manner? The circular plasmid should be cut ideally only once. It does not matter where. But as soon as it is linearized, it should not be ...
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2 answers
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Why does plasmid recombination require precise "copying and pasting" but microinjection doesn't?

Just learning about biotechnological techniques of gene transfer at the moment. With bacterial plasmid recombination, from a high-school level, we are taught that: The desired gene is cut using a ...
4 votes
1 answer
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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 ...
1 vote
1 answer
55 views

What is a "marker-matched" plasmid?

I see this term used a lot in papers and have trouble understanding it. From what I have gleaned it seems they are used during control experiments, perhaps with the goal of making the control plasmids ...
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1 answer
293 views

Function of SMAR in plasmids?

A few years ago, the Thought Emporium published a video (https://www.youtube.com/watch?v=aoczYXJeMY4) in which he refers to a study in which they mix plasmid DNA with Chitosan and feed it to mice to ...
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Can features of modified plasmids be divided into prokaryotic features and eukaryotic features?

Here's what I understand and please correct me if I am wrong: Plasmids modified for gene therapy or genetic engineering should contain factors for certain functions in prokaryotic cells. For example, ...
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Does gene orientation 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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1 answer
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In traditional plant cloning, why do we require two different vectors (plasmids)?

So I was recently taught cloning in plants and I came to wonder what is the need to first put the gene of interest in the entry vector plasmid and then the final vector plasmid before finally ...
3 votes
1 answer
441 views

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

When linearising a vector by restriction digest within the middle of a homologous region can a single cross over integration event only occur if the plasmid is re-ligated within the cell after ...
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181 views

Cloning in a large DNA fragment into a plasmid

I need to clone in a 30kb DNA fragment into my plasmid (~7kb). Working with such a large fragment I have run into a few problems. The first was purifying it from the PCR mix used to amplify it out of ...
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1 answer
173 views

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

I would like to use this plasmid (mammalian expression Flag-HA-USP53 (Plasmid #22606) - Addgene) to produce plasmid in e coli to purify plasmid. Would this work? I am starting out with this vector ...
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1 answer
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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? [closed]

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 ...
5 votes
1 answer
509 views

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 ...
3 votes
1 answer
86 views

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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2 answers
125 views

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 ...
2 votes
2 answers
109 views

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 ...
5 votes
1 answer
158 views

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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1 answer
229 views

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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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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1 answer
128 views

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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1 answer
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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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1 answer
946 views

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.
6 votes
1 answer
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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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2 answers
133 views

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? ...
4 votes
1 answer
263 views

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 ...
2 votes
1 answer
199 views

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 ...
1 vote
1 answer
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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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2 answers
133 views

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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1 answer
157 views

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!...
8 votes
1 answer
18k 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 ...
3 votes
2 answers
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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 ...