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Is PCR used before Molecular Cloning?

Anonymous Wed 20 Nov 19:05:04 2019 No.11163707 View Reply Original Report

Quoted By: >>11163927

PCR cuts DNA of a gene and multiplies a certain gene. DNA Recombinant is putting a gene in plasmid dna and than putting the recombinant plasmid in the bacteria en let the bacteria multiply (clone) and/or produce product of interest/protein .

Is Molecular Cloning isn't done with one plasmid right? so don't you first have to do PCR to cut and multiply the gene of interest, put it into a lot of plasmids and introduce those lot's of plasmids to lots of prokaryotes?

(pic is me trying to be as ugly as possible)

Anonymous

Anonymous Wed 20 Nov 2019 20:23:14 No.11163927 Report

Quoted By:

>>11163707
You're describing two distinct amplification techniques. PCR makes copies of a given DNA strand ex-vivo, while molecular cloning uses fast replicating organisms to make copies of a given DNA strand in-vivo. PCR does not necessarily precede molecular cloning, if that's what you're asking. But more importantly, what is the point of this amplification? The end result often dictates the best path. If you just want to amplify a genome for recognition or genome sequencing, you go for PCR. If you want to have mutant bacteria displaying non innate abilities, synthesising an unnatural compound or simply create a self perpetuating, self replicating, easily spread and extremely cheap genomic library, you go for molecular cloning.

Imagine I want to take a soil sample, destroy the cells, extract the chromatin and run a PCR on the DNA with a fungi specific primer to assess the presence and proliferation of fungi in the soil. PCR is adequate. Remember PCR uses primers, DNA taqpol or Pfupol and lots of dNTPs as building blocks.
Now suppose I just want to make a penicillin resistance gene numbered in the billions. Do I really need a thermocycler, high efficiency DNApol and dNTPs? No, I just need some cheap petri dishes, culture medium and let Nature do its thing.

Anonymous

Anonymous Wed 20 Nov 2019 20:35:22 No.11163960 Report

Quoted By:

What is your end goal? From what you've written I think you trying to express a gene in a bacteria. Here's a brief step by step to do so.

1- PCR the gene of interest
2- Obtain expression plasmid for the bacteria you are going to express it in.
3- Use restriction enzymes to cut open the plasmid and cut the PCR fragment in a manner that the fragment will "fit" in the cut plasmid. I.e. complementary sticky ends.
4- Use ligase to lock the PCR fragment in place.
5- Transform your bacteria with your plasmid. Plate onto suitable selection for your plasmid.
6- Confirm your insert worked by extracting the plasmid and doing a pcr on the plasmid where the insert should be. You can sequence it if you'd like but length should be enough.

Anonymous

Anonymous Wed 20 Nov 2019 21:13:49 No.11164055 Report

Quoted By:

PCR is used at all steps of a cloning project

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