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).
UNAM Genomics Mexico 2011
