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12

This is a common misconception: in fact, in the alkaline lysis method for plasmid isolation, the glucose does not act as an osmotic stabiliser. Glucose is present in the resuspension buffer at 50 mM. This concentration is not high enough for osmotic protection of spheroplasts formed by lysozyme treatment: from memory, 0.5 M glucose is typically used for ...


11

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 ...


9

I like this question, and I had a similar thought when reading that "Forward or Reverse Strand" post. I've since found a 2005 publication by Mirkin and Mirkin1, which investigates the interaction between the replication fork and transcription machinery in context of an E. coli plasmid. An excerpt from the abstract: Studying Escherichia coli ...


9

A plasmid is a length of circular or linear double stranded DNA that exists independent of chromosomal DNA with a cell (i.e. extra-chromosomal DNA) and often confers a selective advantage to an organism such as antibiotic resistance. Plasmids have been identified in archaea, prokaryotes, and eukaryotes and allow horizontal gene transfer within a population, ...


8

If you transfect cells with plasmid then these plasmids need to go into the nucleus (otherwise they wouldn't be transcribed). Getting the plasmid either happens during cell division (when the nucleus is not present) or by adding a signal sequence to the plasmid which induces the import into the nucleus through nuclear pores. The SV40 sequence is an example ...


7

I've found a nice review that has many details on plasmid replication in general, and several papers about pSC101 in detail, and I'll try to extract the key information from these papers. First of all your plasmid as an ori region that contains so called iteron: In many cases, the origin of replication contains directly repeated sequences, termed iterons,...


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 ...


6

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 ...


6

The two plasmids have to have different origins of replication. Two plasmids from the same incompatibility group cannot survive together in the same cell. This quote is from the Wikipedia entry on plasmids: Plasmids can also be classified into incompatibility group. A microbe can harbour different types of plasmids, however, different plasmids can only ...


6

The bacterial cell wall is quite porous, and is not considered a permeability barrier for most small molecules. It mainly functions as structural support and to resist turgor pressures. The average pore size of relaxed peptidoglycans were measured to have a radius in a range of about 2.0 to 2.5 nm, regardless of thickness (i.e. Gram + and Gram - bacteria ...


5

Double transformation (you may want to try searches using that term) has been doable for a very long time, but the efficiency will of course be less than either single transformation below the saturation point. As for your second question, if the cells are made competent, I think a sequential transformation should work.


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


5

In my experience it does not matter if you use water, TBE or TE buffer. Even with a little EDTA in it, it will not mess up enzymatic reactions as the concentration is way too low. TBE/TE buffer usually contains 1mM of EDTA which can chelate 1mM of bivalent cations. Since you not use the DNA undiluted (which also binds a lot of magnesium needed for most ...


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

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

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 you ...


4

You probably mean genomic and non-genomic DNA - Plasmids are small rings of non-genomic double-stranded DNA in Bacteria. They replicate mostly indepently of the genomic DNA can can occur from a few to several hundred copies per cell. See the illustration from the Wikipedia:


4

Purification and isolation of DNA bands by cutting them from agarose gel is commonplace (Lee et al., 2012). The purification step after excision of the band gets rid of most of the EtBr and other impurities. E.g., see the websites from Isogen and Sigma-Aldrich. Reference - Lee et al. J Vis Exp (2012); 62: e3923


4

The ability of bacteria to take up intact environmental DNA is called natural competence. One problem with trying to take advantage of this in a therapy is that it is not very efficient. Importantly, natural competence is regulated and tends to be activated when bacteria are already stressed. This is also likely part of the answer as to why a bacterium ...


4

No, you cannot pellet dissolved DNA with ultracentrifugation. Yes, you can recover a pellet with additional treatments, similarly to how you got it in the first place; only instead of your input being homogenized cells, or tissue, or extract, or whatever you used to obtain your DNA, it would now just be your aqueous solution of DNA. For instance, you may ...


4

Your strategy would work, but if possible you might be better off using XmaI rather than SmaI, as the latter is a blunt-end cutting enzyme. Furthermore, your strategy would not replace the entire gpdA promoter, if that's what you're after. You might wanna refer to their paper, where it's clear from Fig. 1 that the promoter region is much longer than the ...


4

The answer to this is... some. You only need a tiny amount of plasmid DNA to transform bacteria like DH5$\alpha$, typically in the 1-10 ng range, but much less, as low as about 10 pg (10 picograms) may still work. If you can get even 0.2 ul of the liquid out of that tube, try using that in the transformation. Things you can try; remove the paper, spin the ...


4

The GtCCR rhodopsin domain is shown by the thin brown and green arrows on the right of the image that overlap with the EYFP domain and are flanked by the CMV promoter and bGH poly(A) signal. These arrows show the coding region. The sequences for the plasmid are automatically annotated by Addgene with common features (I think they use Snapgene software for ...


3

Plasmapper Is quite good a recognizing common origins of replication. You will have to look up compatibility yourself. It is what I usually use when confronted with unnannotated plasmids.


3

I am sure you have done it; just a note to others: do a PCR screen to ascertain that the plasmid is there. For minipreps you generally do not need a starter culture. If you have screened your clones and now just want to amplify and keep the plasmid for cloning purposes then go for a maxiprep with 200-400ml cultures. Do not overgrow the cells, your ...


3

I'll have to disagree with Alan. If the plasmid is integrated into a chromosome it doesn't lose its episomal status. Actually the integration of the plasmid is a characteristic of an episome. This is a quote from this article: http://www.qub.ac.uk/mlpage/courses/level3/plasmidhistoryreview.pdf "... For a decade or more it was confused with ‘‘episome,’’ ...


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

PREFACE: There's a text, C. elegans: Methods and Applications, specifically doi: 10.1385/1-59745-151-7:109. They outline this exact experiment in it's essence, and one of the diagrams (page 110) notes that for the purposes of RNAi in C. elegans, the T3 promoter specifically doesn't work for the bacterial feeding method, but works for injection or soaking ...


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