Tag Info

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

(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 ...

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 ...

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). ...

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 ...

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 ...

I remember doing this experiment many years ago in an undergraduate practical where we used vigorous vortexing of culture samples in glass tubes to achieve interruption and separation. According to Griffiths AJF, Gelbart WM, Miller JH, et al. Modern Genetic Analysis. Bacterial Conjugation: ... sampling is accomplished by using a kitchen blender to ...

I will also strongly recommend blotting a microgram of your plasmid DNA on a piece of filter paper (the filter paper is important for extraction on the receiving end). Also, it is very very helpful if you clearly indicate the amount of DNA (approximately) and circle the blotted DNA in pen so there is no ambiguity about cutting out the circle of filter paper ...