Molecular Biology Question

[letaps]

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Originally Posted by 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)

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

 

[Phlame217]

I just got through doing this all summer...

You do PCR to amplify the DNA to obtain a sample size large enoguh to get a statistical significance enough to be sure you have the correct DNA. It takes alot of DNA to do this and PCR is the fastest way.

Ligation is done after using the restriction enzymes to break the plasmid open to insert your DNA.

O/N is "over night" typically meaning letting it grow in a nutrient bath over night.

 

[letaps]

Does the process outlined in the first post look correct?

 

[Phlame217]

yes and no, why would you need this tho?

 

[letaps]

what is the wrong with the process?

 

[Vihsadas]

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