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Let's say you want to do a gene cloning experiment. You have an empty expression vector (mRNA or DNA) and are looking to clone some gene X into it. Why is it you can only buy X already inside a vector (which is in turn often inside a bacterial carrier)?

What is the problem with handling pure mRNA nucleotides of gene X? Or even sscDNA?

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    $\begingroup$ Plasmids are vectors. The inserted gene is called a clone or simply an insert that the vector carries., $\endgroup$ – WYSIWYG Mar 15 '16 at 10:13
  • $\begingroup$ @WYSIWYG No, vectors are vectors, plasmids are plasmids, in some cases plasmids are indeed vectors. But literally a plasmid is just a piece of DNA. $\endgroup$ – jiggunjer Mar 15 '16 at 10:25
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    $\begingroup$ I meant the plasmids used in molecular biology, not the natural plasmids. $\endgroup$ – WYSIWYG Mar 15 '16 at 10:27
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What is the problem with handling pure mRNA nucleotides of gene X? Or even sscDNA?

Problems in directly transfecting a mRNA/ssDNA

mRNAs can be transfected into the cells and is quite frequently done. The problem with transfecting mRNAs directly is that the mRNA gets degraded soon and you will not have sustained expression. This is, however, very useful in certain cases such as creating induced pleuripotent stem cells (iPSCs) where you do not want the cells to continually express the Yamanaka factors (some of which are also oncogenes).

Also, RNAs as such are quite susceptible to degradation. It is much easier to prepare and store DNA solutions for a long time.

Similar problems arise with a ssDNA.

Why is it you can only buy X already inside a vector (which is in turn often inside a bacterial carrier)?

One great advantage with using plasmids is that you can propagate your gene infinitely. If you were instead given a fixed amount of mRNA, then you will run out of it soon. Some vendors do practice such greedy capitalistic policies :P

When you are amplifying a DNA using PCR then cloning it has an additional advantages:

  • you can sequence the gene using well standardized primers (like T7)
  • you can make constructs that can express the gene conditionally

You have an empty expression vector (mRNA or DNA) and are looking to clone some gene X into it.

You cannot easily clone something inside an RNA. You need specific RNA endonucleases. Moreover a double digestion will be disastrous in this case.

Why vendors don't generally sell a dsDNA insert but rather provide it cloned in a plasmid

  • A circular dsDNA is more stable
  • Vendors can keep the stock of the insert for future customers without having to do PCR again
  • Cloned insert can be amplified via bacterial cultures to obtain very high amounts (maxi/giga preps)
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  • $\begingroup$ I was thinking from a workflow perspective. When you buy a plasmid (usually double stranded DNA in a bacteria) you need to extract the plasmid first, then cut out the ORF, then amplify and ligate the ORF extract to your own vector. Why don't they just sell vials of ORF? That should be easy to self-propagate too. $\endgroup$ – jiggunjer Mar 15 '16 at 10:51
  • $\begingroup$ @jiggunjer No you generally don't buy the bacterial culture carrying the plasmid. You just get the plasmid dissolved in Tris-Cl pH 7.5. Linear DNA containing just the ORFs cannot self propagate. You can PCR a piece of DNA but with bacterial cultures you can get a lot of DNA (maxi/giga preps). Circular DNA is relatively more stable too. $\endgroup$ – WYSIWYG Mar 15 '16 at 10:56
  • $\begingroup$ Even if one gets pure plasmid, why not pure ORF. I admit the "non-circular is less stable" argument sounds pretty convincing. I know they can't self propagate, but that's why I mentioned the empty vector. So just buy ORF, PCR, and ligate. I'm not very experienced in the lab but I'm trying to get the hang of the protocol, it seems needlessly laborious. $\endgroup$ – jiggunjer Mar 15 '16 at 11:07
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    $\begingroup$ @jiggunjer You do get an empty vector without any insert. Some vendors also sell a pre-digested/linearized vector. $\endgroup$ – WYSIWYG Mar 15 '16 at 11:56
  • $\begingroup$ @jiggunjer Vendors don't sell pre-digested inserts for the reason that there is no one standard vector. Different vectors have different expression sites, which require different restriction enzymes to clone. Selling ORFs would mean the vendor would need to keep stock for hundreds of different kinds of sticky end / recombinase end combinations. It's much easier for the end user to PCR the fragment out and subclone it. $\endgroup$ – March Ho Mar 15 '16 at 12:29

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