12
votes
Accepted
What is the role of glucose in plasmid isolation?
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. ...
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10
votes
Does gene orientation relative to origin of replication matter on small plasmids?
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 ...
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9
votes
Accepted
Is circular DNA the same as plasmids?
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 ...
- 106
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 ...
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5
votes
Accepted
Can I use TBE to elute a plasmid shipped on filter paper?
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 ...
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5
votes
Plasmids for high school students?
I'd suggest looking at the Terms and Conditions for nonprofit use, here is a screenshot of where it is on the webpage:
Look at this form that they link to for legal understanding of what they mean:
...
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4
votes
Can bacteria pick up lethal plasmids?
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, ...
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4
votes
Accepted
Can I pellet DNA back from dissolved state?
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 ...
- 6,192
4
votes
Accepted
How to replace intron region in a plasmid?
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 ...
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4
votes
Accepted
What volume of elution buffer for plasmid-containing filter paper?
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) ...
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4
votes
How do you read the gene insert domains on a plasmid from Addgene?
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 ...
- 11.2k
4
votes
can mammalian expression vector be used in e coli to produce plasmid
Short answer: Looking at the plasmid map, yes, this should work.
Long answer: It depends on the plasmid. To be able to replicate a plasmid in bacteria, you need a origin of replication (ori). To find ...
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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 ...
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4
votes
Function of SMAR in plasmids?
S/MAR (nuclear scaffold/matrix attachment region) Is a DNA sequence pattern that recruits the nuclear protein hnRNP-U/SAF-A a multifunctional scaffold/matrix specific factor.
Jenke et al. 2002
...
- 1,769
3
votes
Is "TATA signal" synonymous with "TATA box"?
"Box" is an archaic term from long ago. It's little used. "Signal" has now replaced it. "TATA box" and "TATA signal" are synonymous.
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3
votes
What, if any, is the difference between covalently closed circular DNA and plasmids?
As a descriptive term, we can take covalently closed, circular DNA (cccDNA) at face value: any DNA that is circular, with the ends joined by covalent bonds, can be described as cccDNA. Thus, plasmids ...
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3
votes
Accepted
When should you use a stringent plasmid?
The three general advantages are:
Lower expression. If your plasmid is expressing something toxic to cells at high levels, reducing the amount of DNA can reduce expression.
Lower plasmid burden. If ...
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3
votes
Plasmid choosing
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 ...
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3
votes
Accepted
Do all RNA polymerase in Eukaryotes share the same transcription factors?
Except for the TATA-binding protein (TBP), there are no common transcription factors for each RNA polymerase. However, there are homologies between many of the factors used by the different enzymes.
...
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3
votes
Large plasmid ligation: do I need alkaline phosphatase?
A plasmid vector will always be prone to recircularisation when a single restriction enzyme has been used, but recombinant plasmids will also be formed during the ligation. Alkaline phosphatase ...
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3
votes
Are my plasmids single-stranded?
You assume that the undigested plasmid is supercoiled, but more probably it is in the relaxed circular form due to single strand nicks. This form migrates slower than linear DNA.
- 17.6k
3
votes
Can bacteria pick up lethal plasmids?
This also lead to the question of whether plasmids can be lethal?
Yes, genes in plasmids could be beneficial, neutral or even lethal, although lethal plasmids may have trouble surviving for long ...
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3
votes
Accepted
Do most bacteria have plasmids?
I think there might be some confusion between individual bacterial cells and "Bacteria" in general. The link you shared is either oversimplifying to the point of losing touch with the truth, ...
- 4,671
3
votes
Accepted
What does Δcys mean after a gene name?
When describing mutations, $\Delta$ usually stands for deleted sequences. In this case it means the exchange of 4 cysteine residues in the NCAM protein by serines to disable palmitoylation and the ...
- 51.5k
3
votes
Accepted
Identifying Promoters in Non-homologous Sequences within the Same Organism
Why does the same promoter within the same organism have such varied sequences?
While there may be other reasons, the fact that the same species has several genes encoding GPD proteins is an ...
- 628
3
votes
Accepted
T-vector creation
Personally I would use Taq DNA polymerase to A overhang my inserts, especially if you are PCR amplifying the insert. It leaves an A overhang on the end of all amplicons natively. All you have to do ...
- 11.2k
3
votes
Accepted
What is a "marker-matched" plasmid?
It is where a control plasmid is used - this is usually an empty vector or a non-target vector with the same "marker" on the plasmid.
The marker is often one that is either used for ...
- 11.2k
3
votes
Cut plasmids with unknown sequence only once
I was going to write a facetious comment asking if the poster believed in fairies, but I restrained myself. Why? 1. because it would not have been nice, 2. because it occurred to me that there was a ...
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3
votes
Linearising plasmids for CRISPR experiment
No. Plasmids should generally be considered as a circular DNA rather than linear. However, no matter where you cut it, the positions don't change.
The position of the cut site is still the position on ...
- 11.2k
2
votes
Question about cutting gene in Plasmid
Following the link you provided confirms that the C. elegans expression vector will express your insert of choice in the transgenic animals' pharyngeal muscle cells (driven by the myo-3 promoter).
...
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