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14

A quite safe way of shipping plasmids is to put them on filter paper (see protocol and send a letter. Much cheaper.


12

Did they try to centrifuge the tube when it got there to push all the liquid to the bottom? I know that especially when working with such little amounts that even shaking it up a little can disperse the contents all over the tube. We have received plasmids from other labs before. Generally speaking the plasmids are sent in Screw-cap microcentrifuge tubes ...


9

Summary the 10 uL of plasmid miniprep may have been splattered in the cap of the tube (AnnaF) the eppendorf tube may have depressurized during air shipment and allowed the 10 uL to escape and evaporate solution: try air-drying or blotting (Jonas) your minipreps prior to air shipment Details As AnnaF wrote, the 10 uL of your plasmid could have been ...


7

In short, yes, the definitions are still correct: The number of copies of a plasmid in the cell is determined by the mechanism of its replication: whether it is synchronized with the replication of the bacterial chromosome or is independent of it. In the first case, the initiation of replication is performed by the same mechanisms of replication of the ...


7

I don't have hands on it, but I will not be surprised if supercoiled DNA migrates at different distances according to some inner topological conformation (i.e., more or less coiled AND/OR more or less nicked). Similarly, this picture highlights >8 conformations. What is run in the gel is circularized phage DNA with some degree of knotting due to the ...


7

Not sure if generalized plasmid taxonomy is going to be relevant any longer. A lot of these old names were created before the exact sequence and function of the various DNA sequences were known. This is becoming especially true as synthetic biology allows us to mix and match parts of plasmids at will. If you want to dig through a lot of the old plasmid ...


6

It seems that no good map of this plasmid is around. Life technology uses it in some of its bacterial strains, the quote: E. coli also contain the helper plasmid, pMON7124 (13.2 kb), which encodes the transposase and confers resistance to tetracycline. The helper plasmid provides the Tn7 transposition function in trans. They link to the original ...


5

Since tubes can be crushed in the mail, the safest way to ship plasmids is to drip a few uL into a filter paper, and then wrap it up to seal it with parafilm, and fill out a detailed form about its content. Good luck.


5

A) Here is the correct map: You made a mistake on your map at the PvuII site (it is not on 6.5kB from the start of the plasmid, but on 6kB). Can the Kpn I not go on the 8.5 site, it still creates the 2 and 8.5, so isn't there more than 1 correct option for plasmid map? Yes. What you need to do in order to make the correct map is try all possible ...


5

I stumbled across this paper demonstrating it is between 150-200 base pairs. DNA flexibility studied by covalent closure of short fragments into circles D Shore, J Langowski, and R L Baldwin PNAS 1981 78 (8) 4833-4837 http://www.pnas.org/content/78/8/4833.full.pdf


4

As can be inferred from the Shanks et al. paper linked in the question: In molecular biology, "suicide plasmid" is a term that refers to a plasmid which is replication incompetent.* Plasmids normally bear a sequence called "origin of replication" Ori which marks the plasmid for replication by the host cell. Plasmids that lack this Ori will not be replicated ...


4

If the plasmids you wish to transform are very big (more than 20kb), the co-transformation might not be very efficient (you might not get any colonies). Electroporation should work though, as electrocompetent cells are more efficient than chemically competent cells. Alternatively, as you have read, you could transform your first plasmid and select for it, ...


4

What I would usually do is transform E. coli with the plasmid, grow an overnight culture and mini prep the plasmid in the morning. Before you do the miniprep save a glycerol stock. Thus, you would have some plasmid to work with in the next couple of weeks, and also a glycerol stock to come back to. You can also keep the plate with your transformants, as you ...


4

We have gotten into similar situations when other labs have sent us plasmids (or when we have taken out ancient tubes out from storage boxes at -20), and have since adopted the filter paper method. Another point to note is that you can just add, say, 10 μL of water to the "empty" tube (Mac) and use 1 μL for a transformation. It has always worked for ...


4

There are a few critical steps (which sounds horrible if written together, but the method per se is robust and usually without problems): Resuspension of the pellet: Make sure it is really resuspended and not floating around as a big blob. If it is not resuspended properly your yields will go down dramatically. NaOH/SDS-Lysis: Don't lyse for too long as ...


3

Gianpaolo is correct that you are seeing the different conformations of circular DNA. Growing plasmids up in bacteria produces 3 main forms: relaxed, coiled and super-coiled. Relaxed tends to run a little higher than the expected size because it cannot travel as efficiently through the agarase; Coiled migrates a little farther than the expected size ...


3

In plasmids with high number of copies, the plasmid needs a certain density to be able to supply its function. If a given cell contains a few dozens of plasmid mollecules, and this is its optimal density, the easiest way to achieve this is to downregulate the replication by its own presence or the presence of one of its components. This also allows a random ...


3

Could be insert polymerization. If you have your stretch of DNA like this: (5')-AATTagctagcatcgtgatcgacg-(3') |||||||||||||||||||| (3')-tcgatcgtagcactagcagcGGCC-(5') And you take that, flip it around, it will ligate onto itself, like this: (5')-AATTagctagcatcgtgatcgacg-(3')(5')-CCGGcgacgatcacgatgctagct-(3') ...


3

(Reposting my comment as an answer since it seems to be what was required.) A DNA molecule that replicates independently of chromosomal DNA is an episome. By this definition a plasmid is (usually) an episome. If a plasmid integrates into a chromosome by some mechanism (as for example in Hfr strains of E. coli where the F plasmid is integrated) the plasmid ...


2

It turns out that there doesn't seem to be a specific mechanism to prevent multiple incompatible plasmids from coexisting. Velappan et al put in more than one incompatible plasmid with different selection genes in them and put the bacteria on a single antibiotic and periodically checked for the second vector (by testing for second antibiotic resistance). ...


2

Although crossover events can be observed in mitosis (mitotic recombination), they most frequently occur in prophase I of meiosis (crossing over in bivalents). Circular chromosomes are common in prokaryotes, but eukaryotes have linear chromosomes. Remember what meiosis accomplishes and that prokaryotes reproduce asexually. Prokaryotes don't have a need for ...


2

Here is a good overview of primer design considerations when using Gibson assembly. http://j5.jbei.org/j5manual/pages/22.html


2

Disclaimer: I haven't ever done a Gibson assembly, but here is my theoretical understanding of how to design your primers. You need four 40mers each consisting of 20 bp segments derived from the vector and the insert and corresponding to the junctions that you are trying to create. In the diagram below the dotted lines represent the junctions between the two ...


2

There are many proposals for the ecological role of restriction-modification (RM) systems, and why they would exist on mobile genetic elements (e.g. plasmids and viruses). In this case, I am specifically talking about the viruses that infect bacteria (aka, bacteriophage). 1) RM systems may have an anti-viral function. Normally we would think of such systems ...


2

The familiar restriction enzyme EcoRI is plasmid encoded. Betlach et al. (1976) A restriction endonuclease analysis of the bacterial plasmid controlling the EcoRI restriction and modification of DNA. Fed. Proc. 35:2037 - 43. For an example of a phage encoded system see: Dempsey et al. (2005) Sau421, a BcgI-like restriction-modification system ...


2

If you have a dsDNA genome and replicate it by rolling circle like most bacteriophages and many plasmids do, an endonuclease is pretty much a necessity.


2

The traditional blue/white screening is set up so that blue colonies are considered positive for the insert, and white colonies are negative. The gene responsible is the lacZ gene, or beta-galactosidase. This enzyme converts a synthetic substrate, X-gal, into an insoluble blue compound. The pCR2.1 TOPO plasmid is a blue/white selection plasmid in which ...


2

It would depend of the plasmid, the genes it contains and the promotors those genes have. Basically, regulation of extrachromosomal genes follow the same patterns of the rest. Bacteria, Archaea and some other organisms that can have plasmids will express the genes if the promotors of those genes are constitutive, or if they trigger by conditions that are ...


2

The strains that we use in the lab have defective restriction machinery. In the case of DH5α it is mostly because of the endA1 and hsdR17 mutation. The former mutation eliminates an endonuclease that can degrade plasmids and the latter one eliminates the restriction system. Check this.


1

pBR322 is a distant ancestor of pET28a. If you compare the two maps below you will see that they have the same replicon labelled as ori or pBR322_origin. The map of pBR322 is shown in the conventional orientation: the single EcoRI site is at coordinate 0/4359 and numbering proceeds in a clockwise direction through the BamHI site. The map of pET28a is ...



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