Questions tagged [plasmids]

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

Filter by
Sorted by
Tagged with
1
vote
1answer
141 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 ...
1
vote
0answers
37 views

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 ...
0
votes
1answer
128 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 ...
1
vote
2answers
981 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 ...
5
votes
4answers
701 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 ...
0
votes
2answers
152 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–...
1
vote
0answers
102 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....
2
votes
0answers
65 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 ...
1
vote
1answer
185 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.
3
votes
1answer
370 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 ...
1
vote
1answer
218 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 ...
5
votes
1answer
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?
1
vote
1answer
248 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 ...
1
vote
2answers
152 views

Plasmid addiction system in high-copy number plasmids

Are there high-copy number plasmids that utilize a plasmid-addiction system?
1
vote
1answer
114 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,...
1
vote
1answer
116 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 ...
3
votes
1answer
236 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, ...
2
votes
0answers
344 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.
1
vote
1answer
100 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. ...
1
vote
1answer
395 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 ...
6
votes
1answer
557 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 ...
5
votes
2answers
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 ...
2
votes
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?
4
votes
1answer
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 ...
3
votes
4answers
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 ...
4
votes
1answer
186 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 ...
9
votes
1answer
763 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 ...
4
votes
1answer
1k 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 ...
4
votes
1answer
372 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 ...
6
votes
1answer
1k 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 ...
1
vote
1answer
424 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 ...
0
votes
1answer
114 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.
1
vote
2answers
3k 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 ...
3
votes
0answers
61 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'...
3
votes
1answer
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?
5
votes
1answer
1k 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.
2
votes
1answer
2k 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 ...
8
votes
1answer
17k 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
3answers
239 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 ...
4
votes
1answer
27k 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-...
5
votes
1answer
762 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 ...
5
votes
1answer
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?
1
vote
1answer
171 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 ...
4
votes
1answer
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 ...
1
vote
1answer
594 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 ...
1
vote
1answer
132 views

How do plasmids protect themselves against restriction enzymes?

How do plasmids protect themselves from restriction enzymes released by bacteria (i.e., against bacteriophages)?
4
votes
2answers
272 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 ...
5
votes
2answers
911 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 ...
4
votes
1answer
364 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 ...
2
votes
2answers
4k 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 ...