103
votes
Accepted
Is a butterfly one or two animals?
There was a paper by: Donald I. Williamson, published in the Proceedings of the National Academy of Science (PNAS) August 28, 2009 https://doi.org/10.1073/pnas.0908357106, and communicated by Lynn ...
- 7,703
6
votes
Accepted
How does the DNA cross through bacterial cell wall during electroporation?
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 ...
- 4,787
6
votes
Plating 96-well bacterial transformations
I think your approach very much depends on how essential it is that you get discrete single colonies, and if this is a one off or a regular process. To preface, similar to what Maximilian Press said, ...
- 61
4
votes
Plasmid Design and Integration events [Single vs Double cross over]
Single crossover is not the result of religation in the cell. The double-stranded break (DSB) in the plasmid instead stimulates a homologous recombination event, causing the entire plasmid to ...
- 638
3
votes
I accidentally plated DH5a E. coli and left them in the 4℃ for 12 hours, if I put them into the 37℃ incubator will they start growing?
If these were just regular E. coli that you were growing up, then it wouldn't matter too much - they would certainly grow a lot slower on the plate than you might otherwise expect compared to a plate ...
- 12.9k
3
votes
are there any species that we can identify as being 'mid-way'though an evolutionary change?
Every species in existence is "midway through its evolution"
This is because evolution is a never ending process. Technically, we humans are still evolving. Evolution only ends when the species dies ...
- 207
2
votes
How to prevent a old promoter region from attaching onto a plasmid instead of a new one during ligation?
Run it out on a gel after digestion and extract the vector only.
Treat with a phosphatase after digestion. This will prevent religation since you need at least one of your fragments to be ...
- 747
2
votes
In traditional plant cloning, why do we require two different vectors (plasmids)?
If you mean the binary system using the Ti plasmid:
The main issue is that on the massive Ti plasmid there is a difficulty locating unique restriction sites (see section "Introduction of Genes ...
- 41
2
votes
Which method of gene amplification for toehold switches?
I basically agree with Bob, but wanted to add a few more details.
Out of the options that you have available, option 4 is the most common way to do it. I wouldn't consider options 1 and 3. I don't ...
- 634
2
votes
Which method of gene amplification for toehold switches?
Option 2 is your simplest and easiest. It should give you unlimited plasmid to work from. Note that ordering pre-made sequences of this size isn't particularly cheap and might take a few weeks to get ...
- 12.9k
2
votes
Plating 96-well bacterial transformations
I would suggest pouring solid media selection plates of something like this, that have the same shape and size as the 96-well plate. You can then directly spot your transformations from their wells ...
- 10.5k
2
votes
Accepted
How to separate two plasmids from E coli with the same backbone?
There's a couple of options here - it could be that your plasmid extract isn't very pure, so some genomic DNA has come over and the ROI is in that. The PCR would detect this even if you don't have any ...
- 12.9k
1
vote
Terminology for Transgenes
Yes, I would change to a different terminology. I would not apply the word 'allele', which would be confusing/misleading at best. Even if the transgene is an allele taken from an organism, I would ...
- 109
1
vote
Gene of interest number of copies in transformed K. pastoris
To predict the number integrations is probably not possible, but you can favor multiple integrations, if that's what you are after. At least in Saccharomyces cerevisiae integration of a first plasmid ...
- 638
1
vote
Accepted
How to identify the GPD gene when the sequence varies between organisms?
I might be misunderstanding you here, but I take from your links above that you want to match the GPD promoter regions from two (distantly related) fungi Agaricus bisporus and Lentinula edodes, to the ...
- 638
1
vote
How can I improve efficiency of Ecoli transformation?
The plasmid is quite big which might explain why it is difficult to transform bacteria (is it a lentiviral vector?).
If you could give additional details you might have a chance to have improved ...
- 620
1
vote
are there any species that we can identify as being 'mid-way'though an evolutionary change?
No, because that assumes evolution has some "end goal" which of course is not the case.
The statement "surely there are some evolved biological characteristics that are only beneficial once 100% ...
- 106
1
vote
Heat shock vs electroporation
How do you detect a potenially positive transfection? Short fragments might have proper teriary structures which may make them behave differently than you would expect. Also, noncircular DNA and RNA ...
- 21
1
vote
Accepted
Cloning DNA fragment - at least trying to
The fragment size itself shouldn't be a problem in principle.
Cloning has many steps, so anything you didn't specifically test could be causing problems. If different vectors are also not the problem,...
- 4,391
1
vote
Why must gene entrapping constructs be integrated into introns and not exons?
I don't know that they must integrate into an intron. It seems perfectly reasonable that they could also integrate into an exon, as long as the reporter is in-frame. There is obviously an element of ...
- 17.8k
1
vote
LacZ' selection: blue colonies despite ligation of insert
Most inserts will disrupt beta-gal expression (by shifting the reading
frame and introducing stop codons) and therefore remove the enzymatic
activity required to cleave X-gal and produce the blue ...
- 351
1
vote
Viruses and Transformation
The answer lies in the definition itself of transformation. See from here:
In molecular biology, transformation is the genetic alteration of a cell resulting from the direct uptake and ...
- 14.1k
1
vote
Luciferase promoter vector over p-AcGFP1-C1 vector
You are confusing luminescence and fluorescence. GFP does not emit light. The abbreviation stands for Green Fluorescent Protein. You need to shine a blue light source at it and it will fluoresce green....
- 1,425
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