recombinant dna

[yoni]

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anybody who can answer this would be much appreciated.
thought i understood this but now i am very confused. so if you already have plated out the colonies that contain the recombinant plasmid using the antibiotic resistant technique then why is there a need to indentfy the correct clone colony later using specific hybridization, antibody screening, and pcr techniques??? don't you already know which colonies have your dna of interest in the vector (or else they wouldn't grow)? i think this is the start of my confusion.
thanks,
yoni

 

[BubbleWand1]

anybody who can answer this would be much appreciated.
thought i understood this but now i am very confused. so if you already have plated out the colonies that contain the recombinant plasmid using the antibiotic resistant technique then why is there a need to indentfy the correct clone colony later using specific hybridization, antibody screening, and pcr techniques??? don't you already know which colonies have your dna of interest in the vector (or else they wouldn't grow)? i think this is the start of my confusion.
thanks,
yoni

one of the things i can think of is that sometimes if you're trying to clone dna into the colonies, it can go in backwards and by screening, you're going to only pick the colonies that took up the vector correctly - that and if you run into problems later on, by screening you make sure that the colony did in fact havethe insert.

 

[v-tach]

anybody who can answer this would be much appreciated.
thought i understood this but now i am very confused. so if you already have plated out the colonies that contain the recombinant plasmid using the antibiotic resistant technique then why is there a need to indentfy the correct clone colony later using specific hybridization, antibody screening, and pcr techniques??? don't you already know which colonies have your dna of interest in the vector (or else they wouldn't grow)? i think this is the start of my confusion.
thanks,
yoni

Sometimes there will be colonies that took up a vector plasmid which did NOT get the insert DNA, so they will have the antibiotic resistance and therefore grow, but do not have the gene of interest. Hybridization allows you to find the colonies that not only took up a plasmid, but took up a plasmid that contains the gene being studied.

 

[b&ierstiefel]

Hi yoni,

To answer the question you pose, one needs to consider how a DNA construct is made.

Simply, a circular plasmid is digested by a restriction enzyme. The insert is also digested by the same enzyme.
Then the digested plasmid (vector) is ligated to the insert (gene of interest).
Finally, the resulting reaction mixture is used to transform bacteria.

I'm sure you know this...but this sets up what I'm gonna say next.

Now let's think of the possible scenarios given that you can have the following components in your reaction tube after the ligation reaction:
(1) New circular plasmid consisting of the vector ligated to the insert.
(2) Undigested circular vector plasmid
(3) Digested linearized vector plasmid
(4) Undigested insert
(5) Digested insert

Bacteria are transformed by circular plasmids. Linear DNA do NOT make it into bacteria during the transformation. Hence, species (1) & (2) can transform bacteria and confer antibiotic resistance hence leading to eventual colony formation.

See the problem here? If you get a colony, how do you know if the colony harbors (1) which is what you want -OR- (2) which is what you want. To distinguish if the colony is a TRUE positive (1) rather than a FALSE positive (2), ancillary methods are required. This can be done by several methods such as southern blot (in real life, nobody does this to verify true positive clones), PCR, and miniprepping DNA and subsequently digesting it with the same enzymes you used before to verify that the insert is present when you run it on the gel.

Hope that clears things up.

 

[Zuwie]

If you cut the whole DNA with restriction enzymes, there will be many different fragments located between those specific restriction sites. All these fragments will be recombined into plasmids containing antibiotic resistance genes. But you only need to select for bacteria that took in plasmids which incorporated the gene you are interested in.

 

[yoni]

good show, thanks so much.
yoni

 

[Sean2tall]

Not to mention that you could have a contaminated plate, where some other plasmid that you cloned last week is giving the resistance.

Or plain ol' magic.