User:Daniel Ramirez/Notebook/UNAM Genomics Mexico 2011/2011/07/14

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χρόνος πέρασμα July 14th 2011


  • Obtainment of the cDNA from the mRNA of the genes HydEF and HydG from Chlamydomonas reindhartii.

  • Today I made cDNA from the RNA extraction from Chlamydomonas reindhartii Helena Reyes did. In order to make cDNA one can either use special oligonucleotides (dT) that are used as templates by the Reverse transcriptase enzyme and produce a complementary version of all the mRNA in the solution that have been polyadenilated. The procedure I follow consist of using specific oligonucleotides that are complementary only to the coding region of the genes HydEF and HydG from C. reindhartii; this means that only the genes that we wish to obtain will be transformed into cDNA.
  • The oligonucleotides that were sent synthezised were very concentrated, that's way I diluted all of them to a concentration of 10 pmol/μl. The oligos were at an initial concentration of 376.14 pmol/μl, 344.8 pmol/μl, 316.72 pmol/μl and 138.46 pmol/μl for HydG 5' end, HydEF 5' end, HydEF 3' end and HydG 3' end, respectively. I took 13.28 μl from HydG 5' end, 14.5 μl from HydEF 5' end, 15.78 μl from HydEF 3' end and 38.33 μl from HydG 3' end and added enough water to each to reach a volume of 500 μl.
  • I prepared two solutions for each gene (from each of the RNA extraction cleaned from DNA that Helena and I did) to make the cDNA. Each tube contained:

Template RNA 5 μl
3' reverse oligo 2 μl
H20 4.5 μl
Total 11.5 μl

  • All four tubes were put into the thermocycler during 10 minutes at 70°C and then added with the following reactants:

5x Reaction Buffer 4 μl
H20 0.5 μl
dNTP 10mM 2 μl
M-MulV Reverse Transcriptase 2 μl
Total 8.5 μl

  • This 20 μl final solution was left incubating during 56 minutes at 37°C and then 15 minutes at 70°C to deactivate the enzyme. After this, the PCR tubes were left at -20°C.