I'm writing an essay about DNA recombinant techniques where the question asks to outline all the steps to arrive at having a colony of bacteria expressing a gene of interest.The question makes you start with a pool of mRNAs, which I assume is for making cDNA. However, how do you go from having thousands of different mRNAs to just one kind?

  • 1
    $\begingroup$ Your cDNA approach is correct - you use poly-A primers to make cDNA for all mRNAs in the mix, resulting in a mix of cDNAs. Now how do you isolate the one you want from the mix? $\endgroup$
    – Armatus
    Apr 5 '18 at 14:13
  • $\begingroup$ Would you use Southern Blotting on the cDNA? would Northern blotting on the mRNAs bring you to the same end point? $\endgroup$
    – Matt
    Apr 5 '18 at 14:55
  • $\begingroup$ Try reading this product insert (downloads a PDF) from Miltenyi GmbH. Half the battle is knowing what techniques or products exist, and when/how to use them. Granted there are other things you could do. $\endgroup$
    – CKM
    Apr 5 '18 at 15:34

First of all you need to figure out what your goal is: an expression vector (i.e. plasmid) which will drive expression of a gene in bacteria (let's say E.coli), and your gene's coding sequence needs to be downstream of the promoter in that construct.

Your task is asking for a pretty routine lab task known as cloning (https://en.wikipedia.org/wiki/Molecular_cloning). Your mRNA pool might be the result of an RNA extraction from a tissue sample and your goal is to get bacteria that produce lots of the desired protein.

As this is homework, I will only give a broad overview and leave you to figure out the details.

Indeed one way you could approach this is actually some method to specifically extract the desired mRNA, then generating cDNA from it and pasting that into the vector. This is unlikely to give you good success rates, and in practice nobody purifies a specific mRNA unless they are interested in other molecules naturally bound to that mRNA.

Since this is a procedure with a very practical purpose without a scientific question behind it, there's a much more efficient way to achieve it.

  1. Generate cDNA for all mRNAs in the mix ("first-strand cDNA synthesis").
  2. Instead of removing unwanted RNAs, simply amplify the desired one using specific primers you designed and PCR. This gives you what is called the "insert".
  3. Most likely if you ordered the expression vector online, you actually receive bacteria containing it, so you first have to culture those and extract DNA from the culture ("miniprep").
  4. Next step is to paste the DNA into the expression vector. For this you need to digest both the vector and your insert using the same restriction enzymes ("restriction digest"). (Your insert will not have restriction sites on it, so you will need to introduce them by designing the primers for the PCR in step 2 appropriately.)
  5. Run the restriction digest mixes on a gel (electrophoresis), identify the bands corresponding to the insert in one lane and the vector in the other, and extract them (gel extraction).
  6. Ligate the insert and vector
  7. Transform the finished product into competent E.coli

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