Flow Cytometry Technical Resources

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DNA library preparation

If the goal is to create a genomic DNA library, this first step is to extract genomic DNA (please see the protocol for DNA extraction ). Whereas the first step to generate a cDNA library relies on mRNA extraction (please see the protocol for RNA extraction ). Afterward, the mRNA is converted to cDNA through the catalytic activity of reverse transcriptase enzyme (please see the protocol for conversion of mRNA into cDNA).

Once the cDNA is obtained, the use of restriction enzymes is required to create complementary ends in the vector and in the DNA fragments.  

DNA digestion:

  • In 200 μl tube add 2 μl of cDNA or genomic DNA
  • Add 15 μl of DEPC-treated water
  • Add 2 μl of restriction enzyme buffer (10x)
  • Add 1μl of restriction enzyme
  • Incubate for 2h at proper temperature accordingly the restriction enzyme selected
  • Inactivate the restriction enzyme at high temperature (usually 20 min at 65 ºC)

Vector digestion:

  • In 200 μl tube add 2 μl of vector (100 ng/μl)
  • Add 15 μl of DEPC-treated water
  • Add 2 μl of restriction enzyme buffer (10x)
  • Add 1 μl of restriction enzyme
  • Incubate for 2h at proper temperature accordingly the restriction enzyme selected
  • Inactivate the restriction enzyme at high temperature (usually 20 min at 65 ºC)

Cloning:

  • In 200μl tube add 5 μl of digested cDNA
  • Add 3 μl of digested vector
  • Add 1 μl of ligase buffer (10x)
  • Add 1 μl of ligase enzyme
  • Incubate at 4ºC overnight
  • Use 5μl of the ligation solution to transform host competent cells
  • Plate the cells in media with a selection marker, e.g. ampicillin, kanamycin, depending on the selection marker of the vector used.

DNA library screening

To functionally characterize and identify new genes, physiological tests can be carried out to detect phenotypic differences between the host organism and the host organism carrying DNA fragments of the DNA library. Usually growth tests are the most common approach to achieve this task, either by cultivating cells in different media formulations and/or different temperatures.

One real-life example is the use of renewable carbon sources aiming to produce biofuels as a recent initiative to find alternatives to fossil fuels. Most of the organisms used in this bioprocess, like E. coli, are not able to grow in these substrates, namely xylan. But the degradation product of xylan, which is xylose, can be assimilated and metabolized by industrial microbes (and used to produce biofuels).  Thus, samples of microbes growing in substrates rich in xylan, were used to extract the RNA and prepare cDNA libraries as described previously, and to be transformed in E. coli competent cells. The transformed cells are subsequently plated in minimal media with xylan as sole carbon sources. This procedure will allow positive selection of transformants. Only transformants that carry genes in the vector that code for enzymes able to degrade xylan into xylose will grow in the plate. The vector of the transformants is then extracted and sequenced. This will allow us to identify which genes are responsible to allow growth in xylan media.