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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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28 votes
6 answers
14k views

How should I ship plasmids?

I shipped 10 µL 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 ...
Nick T's user avatar
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13 votes
3 answers
22k 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 ...
Ben's user avatar
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11 votes
2 answers
1k 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. ...
Armatus's user avatar
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10 votes
1 answer
5k views

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 ...
Arcadium's user avatar
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9 votes
1 answer
872 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 ...
Neil's user avatar
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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 ...
Superbest's user avatar
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8 votes
1 answer
810 views

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 ...
jakebeal's user avatar
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8 votes
1 answer
295 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 ...
shigeta's user avatar
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6 votes
1 answer
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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 ...
Robertos's user avatar
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6 votes
1 answer
600 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 ...
Varekai's user avatar
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6 votes
1 answer
163 views

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 ...
ceno980's user avatar
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5 votes
2 answers
1k 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 ...
Kelly Eckartt's user avatar
5 votes
4 answers
1k views

Picking up Plasmid DNA using Nanodrop, but not using Electrophoresis

I am currently trying to see if 2 bacteria contains plasmids or not. I had used promega plasmid extraction kit on the bacteria. I ran a gel electrophoresis (.8% gel) with the extract product, however ...
A. Radek Martinez's user avatar
5 votes
1 answer
2k 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.
abc's user avatar
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5 votes
1 answer
2k 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?
Mark B's user avatar
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5 votes
1 answer
510 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 ...
doremi's user avatar
  • 327
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 ...
doremi's user avatar
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5 votes
1 answer
932 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 ...
LanceLafontaine's user avatar
5 votes
2 answers
1k 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 ...
Armatus's user avatar
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5 votes
1 answer
5k 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?
James Hiew's user avatar
4 votes
1 answer
607 views

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.
123321123321's user avatar
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 ...
Treex's user avatar
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4 votes
1 answer
31k 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-...
Superbest's user avatar
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4 votes
2 answers
220 views

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 ...
pstumps's user avatar
  • 55
4 votes
1 answer
5k 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 ...
Will Perry's user avatar
4 votes
2 answers
435 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 ...
biogirl's user avatar
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4 votes
1 answer
94 views

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 ...
Sam's user avatar
  • 41
4 votes
1 answer
3k 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 ...
WilliamL's user avatar
  • 133
4 votes
1 answer
551 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 ...
SpeedOfLight's user avatar
4 votes
1 answer
4k 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 ...
Gergana Vandova's user avatar
4 votes
1 answer
8k views

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 ...
Krishi1234's user avatar
4 votes
1 answer
221 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 ...
user3473083's user avatar
4 votes
1 answer
2k 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 ...
Alice's user avatar
  • 240
4 votes
1 answer
424 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 ...
bsubspore's user avatar
4 votes
1 answer
311 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 ...
nanofarad's user avatar
  • 306
4 votes
0 answers
160 views

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 ...
alec_djinn's user avatar
  • 3,108
3 votes
2 answers
147 views

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 ...
liliae's user avatar
  • 636
3 votes
3 answers
272 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 ...
biogirl's user avatar
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3 votes
2 answers
12k 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 ...
Armacino's user avatar
3 votes
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 ...
projective_line's user avatar
3 votes
1 answer
444 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 ...
Milney's user avatar
  • 31
3 votes
1 answer
213 views

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 ...
Michael's user avatar
  • 31
3 votes
1 answer
2k 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?
frenchwhorne's user avatar
3 votes
1 answer
87 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 ...
Kat's user avatar
  • 33
3 votes
1 answer
265 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, ...
Scsqpd's user avatar
  • 195
3 votes
4 answers
3k 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 ...
March Ho's user avatar
  • 9,452
3 votes
1 answer
2k 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. ...
meldob's user avatar
  • 33
3 votes
1 answer
452 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 ...
user137's user avatar
  • 5,348
3 votes
1 answer
73 views

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). ...
Julius's user avatar
  • 31
3 votes
0 answers
64 views

Mechanistically, how does calcium chloride aid in bacterial transformation? [duplicate]

I am looking to get more information about the mechanism of the $CaCl_2$ in the bacterial transformation. I read online about the topic but I didn't found any reverent information on the topic, I don'...
Matt's user avatar
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