User:Daniel Ramirez/Notebook/UNAM Genomics Mexico 2011/2011/07/15

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χρόνος πέρασμα July 15th 2011


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

  • Today I made a PCR to amplify the cDNA of the genes HydEF and HydG from Chlamydomonas reindhartii I made yesterday. Four PCR tubes with added with the following:

H20 9 μl
Buffer rTth [3.3x] 6 μl
Mg(OAc)2 [25 mM] 3 μl
dNTPs [10 mM] 3 μl
Oligo forward [10 pmol/μl] 2 μl
Oligo reverse [10 pmol/μl] 2 μl
DNA template 5 μl
Total 30 μl

  • After putting the PCR tubes into the thermocycler during 2 minutes at 94°C, the following reactants were added to each:

H20 10.5 μl
Buffer rTth [3.3x] 9 μl
rTth DNApol 0.5 μl
Total 20 μl

  • Finally, the now 50 μl volume tubes where put at 30 cycles consisting of 0:45 minutes at 94°C, followed by 0:45 minutes at 60°C, followed by 3:30 minutes at 72°C; and after the cycles 5 minutes more at 72°C.
  • A 1% agarose electrophoretic gel was made to test if the amplification was succesful. 5 μl of the PCR product was used, along with 3 μl of dye. Well #1 contains 2 μl 1kb DNA ladder, wells #2 and #3 contain PCR amplified with HydG oligos (Mine and Helena's) and wells #4 and #5 contain PCR amplified with HydEF oligos (again, mine and Helena's).

Creindhartii HydG HydEF PCR.jpg

  • There was no amplification :(