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 ...
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
As AnnaF wrote, the 10 uL of your plasmid could have been hidden ...
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 ...
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 ...
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 ...
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 ...
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 ...
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,...
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 ...
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 ...
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
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 ...
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 ...
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.
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.
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 us....
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, ...
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 ...
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 ...
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:
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.
- Lee et al. J Vis Exp (2012); 62: e3923
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 ...
Could be insert polymerization. If you have your stretch of DNA like this:
And you take that, flip it around, it will ligate onto itself, like this:
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,’’ ...
(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 ...
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 ...