Dna extraction, amplification and gel electorphoresis

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username3501624
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#1
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Hello, could someone please help me with the disadvantages/advantages and strength and weaknesses with each one ?
and transformation of cells
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Callicious
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I don't wish to sound rude or anything, but, with reference to what other methods/etc? I mean there are lots of ways to extract DNA/amplify it, but I suppose Gel Electrophoresis could work.

Gel Electrophoresis:
- Relatively slow process: It can be automated, as was done for some projects in the past, however in the end you do still need to allow a good amount of time for propagation of the genetic material through your electrophoretic medium
- Needs quite a large amount of genetic material, i.e. to provide substantial visibility during imaging. Getting this material either requires a large initial sampling, or a good amount of amplification, and dependent on the amplification amount can damage the sample/corrupt it, i.e. through introducing foreign material, copying errors, etc.
- Limited in terms of the size/length of the DNA to be sampled: you could use it to sample section of material or something along those lines, but sampling a piece of genetic material many thousands if not millions of bases long would be pretty impractical with this method, as you'd need more medium and larger setups, or just more time: next generation sequencing methods require less time, material and space in comparison.
however
- It can be more convenient to do with higher availability of material, medium, etc
- It is a lot cheaper to do for short sequences, i.e. for demonstrative purposes
- It's simple and highly understood, thus little room for error
- It's reliable, having been in use for such a long time versus NGS methods.

That's all I've really got for Gel Electrophoresis; for Amplification and/or DNA Extraction you'd need to specify a certain method :-;
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Callicious
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OH also, Transformation of Cells.

Assuming you mean Horizontal Gene Transfer, with transformation being one of the methods behind it, a free bacterium may take up some RNA that it finds roaming in whatever medium it's currently... well, roaming around in; once it takes up this RNA or can integrate it into its own genetic material, i.e. in a plasmid.

In general it's just the uptake of genetic material from the environment and its incorporation.
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(Original post by Callicious)
I don't wish to sound rude or anything, but, with reference to what other methods/etc? I mean there are lots of ways to extract DNA/amplify it, but I suppose Gel Electrophoresis could work.

Gel Electrophoresis:
- Relatively slow process: It can be automated, as was done for some projects in the past, however in the end you do still need to allow a good amount of time for propagation of the genetic material through your electrophoretic medium
- Needs quite a large amount of genetic material, i.e. to provide substantial visibility during imaging. Getting this material either requires a large initial sampling, or a good amount of amplification, and dependent on the amplification amount can damage the sample/corrupt it, i.e. through introducing foreign material, copying errors, etc.
- Limited in terms of the size/length of the DNA to be sampled: you could use it to sample section of material or something along those lines, but sampling a piece of genetic material many thousands if not millions of bases long would be pretty impractical with this method, as you'd need more medium and larger setups, or just more time: next generation sequencing methods require less time, material and space in comparison.
however
- It can be more convenient to do with higher availability of material, medium, etc
- It is a lot cheaper to do for short sequences, i.e. for demonstrative purposes
- It's simple and highly understood, thus little room for error
- It's reliable, having been in use for such a long time versus NGS methods.

That's all I've really got for Gel Electrophoresis; for Amplification and/or DNA Extraction you'd need to specify a certain method :-;
For the DNA extraction the one where you break the cells open to expose the DNA, then adding detergent, putting it in a hot water bath then adding ice cold ethanol (if that helps ? )
And for the amplification im talking about polymerase chain reaction btw thanks for the info about gel electrophoresis
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Callicious
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(Original post by Reann01)
For the DNA extraction the one where you break the cells open to expose the DNA, then adding detergent, putting it in a hot water bath then adding ice cold ethanol (if that helps ? )
And for the amplification im talking about polymerase chain reaction btw thanks for the info about gel electrophoresis
Yo;

Okay, so... I've not done that in a year or so, and I can't really remember all that well for the DNA extraction advantages or disadvantages. Amplification wise, I'm glad to help c;

Okay, so... using TAC Polymerase for the PCA cycle:

- Limitation regarding the number of times you can carry out the process: ~20 is what I remember. Thermocycling through high and low temperatures damages the enzymes, and for some reason or other (literally what it said back then) you will end up with a limit, anyway, due to the enzymes stopping their work.
- Limitation on the amount of amplification due to substrate concentrations: eventually you're going to run low on substrate, and the polymerase can't polymerise without substrates, i.e. nucleobases and all that good stuff.
- A sharp limitation based on the material used. Sadly I can't give you the specifics seeing as I forgot this, but, if you use jeans the dye that most use (i.e. for the blue) inhibits the TAC Polymerase. Older samples, i.e. archeological, also have an issue along these lines.
- TAC Polymerase also has a high error rate. Reducing temperature can increase maximum length of the DNA chains, and using DNA polymerase can increase it further, though this increases time for thermocycling/etc.
- Subject to contamination. A single bit if contaminant from the laboratory technician could instantly be amplified, invalidating the sample.
advantages
- Pretty darned fast. Thermocycling is automatic, and thus the process can be left to function.
- Pretty cheap material wise in comparison to other methods
- Produces large volumes of amplified DNA
- Relatively accurate for small segments of DNA

That's the limit of what I can help, sorry
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