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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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21 views

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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915 views

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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164 views

Why are two forward and reverse priming sites depicted here? what do they do?

This question is in regard to the baculovirus expression system. (source: amsbio.com) Why do we need to generate primers for the polyhedrin promoter and the baculovirus? Primers are needed to ...
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40 views

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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166 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.
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203 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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35 views

Yeast integrating vectors: where to cut?

I understand that yeast integrating plasmids (YIps) must be linearized in order to promote homology-directed recombination. However I don't quite understand where. In the end, the external regions of ...
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Are there vectors available without any promoter?

Looking for a vector with the following characteristics: 1. Without any promoter 2. Selectable marker: Puromycin 3. Blunt end restriction sites 4. Transfection host: Human or Mouse cell lines.
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59 views

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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68 views

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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42 views

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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2k 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 ...
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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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1answer
117 views

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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1answer
4k 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 ...
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1k 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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444 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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2k 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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141 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 ...
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189 views

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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1answer
996 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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146 views

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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1answer
98 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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1answer
98 views

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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2answers
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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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1k views

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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1answer
157 views

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 ...
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1answer
318 views

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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1k views

What, if any, is the difference between covalently closed circular DNA and plasmids? [closed]

Are cccDNA (covalently closed circular DNA) and plasmids the same thing? What about DNA topoisomers?
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310 views

Is “TATA signal” synonymous with “TATA box”?

In the text I'm translating I have a diagram of an expression vector. It has a lot of marks, and one says "TATA signal". I googled and found the expression "TATA box". Are these expressions fully ...
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2answers
3k views

What are the possible reasons to get extra unrecognized band in agarose gel electrophoresis?

I ran 0.7% agarose gel electrophoresis of my plasmid DNA sample consisting of GFP vector, using NheI and HindIII restriction enzyme. I used two types of plasmids, i.e., isolated by miniprep-alkali ...
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What placement of plasmid parts makes the most sense?

If you want to express the largest amount of a certain or a few proteins using a plasmid, does it make sense for the antibiotic resistance gene to come after genes for proteins you intend to ...
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1answer
667 views

Reading annotations from a plasmid map

Can anyone explain why inside plasmid mapping software the annotations are layered under each other, I am confused as to why there are essentially two layers within the map, for example in the image ...
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1answer
137 views

Plasmid specific DNA degradation

I'm interested if there is a way to degrade the plasmid DNA inside an E.Coli cell specifically so that the method does no harm to the chromosomal DNA. First I was thinking about restriction ...
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2answers
3k 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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Questions in Regards to Isolating Promotor Region for Recombinant DNA Techniques

For a while now I have been trying to isolate the promotor region of this Protein. Using sticky end PCR, I need to isolate this promotor region so it has paticular sticky ends for the purpose of ...
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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'...
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894 views

Evolutionary advantage of F plasmid in bacterial conjugation

Hi I would like to ask a question on bacterial DNA exchange, namely conjugation via F plasmid. It seems to me that all the F plasmid codes for is the sex pilus. What is the evolutionary advantage of ...
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Is it possible to both extract DNA sequences from plasmids and fuse them using primers for Gibson assembly?

Maybe I'm overcomplicating this, but I'm having some issues with a construct. So, I have three plasmids containing a total of eight DNA sequences that I need to fuse into one linear sequence. I plan ...
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121 views

Restriction enzyme digestion

A circular plasmid of 10,000 base pairs (bp) is digested with two restriction enzymes, A and B. This produces a 3000 bp and a 2000 bp bands when visualized on an agarose gel. When digested with one ...
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4answers
571 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 ...
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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. ...
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138 views

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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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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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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1answer
362 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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3k 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?