Molecular Biology Question

[letaps]

Hey,

What is purpose of doing a miniprep? And why do we do a DNA digestion after doing a miniprep? What applications do they have?

Does anyone have a good picture explaining DNA digestion (link maybe?)?

Thanks

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[xIcewind]

Miniprep basically isolates the plasmid DNA (through a kit) from a bacteria culture grown from a single colony.

This plasmid is usually (well, hopefully) inserted with the strand of DNA you want in the vector.

Basically. You ligate your DNA (say, it codes for proteins) with two restriction enzyme digests into your plasmid. You transform the plasmid into the bacteria and then plate it, take a colony, and do an O/N culture to harvest the plasmid.

Your DNA digestion (with the same two restriction enzymes) will cut at the site where it's supposed to, and then a gel following will show you if the transformation worked. If it did, you'll see two distinct bands -- your vector and your DNA. If you see one, it's usually an empty vector.

Transformation and miniprepping are the standard techniques to go from DNA (from PCR) to a bacteria colony for expression (i.e. for proteins)

 

[letaps]

Miniprep basically isolates the plasmid DNA (through a kit) from a bacteria culture grown from a single colony.

This plasmid is usually (well, hopefully) inserted with the strand of DNA you want in the vector.

Basically. You ligate your DNA (say, it codes for proteins) with two restriction enzyme digests into your plasmid. You transform the plasmid into the bacteria and then plate it, take a colony, and do an O/N culture to harvest the plasmid.

Your DNA digestion (with the same two restriction enzymes) will cut at the site where it's supposed to, and then a gel following will show you if the transformation worked. If it did, you'll see two distinct bands -- your vector and your DNA. If you see one, it's usually an empty vector.

Transformation and miniprepping are the standard techniques to go from DNA (from PCR) to a bacteria colony for expression (i.e. for proteins)

So, will the process go in this order: reverse transcriptase to turn RNA into cDNA, then do PCR (why would u do PCR here?), then digest the cDNA to see if it has the specific fragment that codes for a certain protein, then another PCR (using specific primers) to obtain the specific fragment, then a miniprep to obtain plasmid from bacteria, then another digestion to add the fragment to the bacteria for expression. Does that look right?

And where does ligation come into play?

What is O/N?

Thanks

 

[xIcewind]

Practically, you'd take the cDNA of the bacteria (or organism from which you want to extract the section of DNA you want), transcribe the section, PCR it (to amplify the # of such sections), ligate it into the vector (so your vector carries the DNA you want), transform vector into bacteria (so your bacteria has this vector), then miniprep bacteria (to check that it wasn't an empty vector that has been transformed).

O/N = overnight.

Haha. I'm bitter about the amount of this stuff I have to do. (Not really -- in a sad, routine way, I find this kind of fun.)

 

[letaps]

Practically, you'd take the cDNA of the bacteria (or organism from which you want to extract the section of DNA you want), transcribe the section, PCR it (to amplify the # of such sections), ligate it into the vector (so your vector carries the DNA you want), transform vector into bacteria (so your bacteria has this vector), then miniprep bacteria (to check that it wasn't an empty vector that has been transformed).

O/N = overnight.

Haha. I'm bitter about the amount of this stuff I have to do. (Not really -- in a sad, routine way, I find this kind of fun.)

oh ok...that helps a lot...lets say that we did a digestion after the miniprep....what would that accomplish?

Thanks

 

[xIcewind]

A digestion after miniprep cuts at the two restriction sites on your vector. If your ligation was successful, then you'd get the DNA band and the vector band. If your ligation failed, then you'd get an empty vector -- just one band, without the DNA band. You'd quantify this, of course, by running an agarose gel.