IGEM:UNAM-Genomics Mexico/2009/Notebook/H2 Pae/2011/08/22

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  • Continuing the assembly of the parts which are going to be sent to the registry.

Parts assembly

Today I did a PCR from the pSB1C3 plasmid amplifying all the backbone, because I will need it to isert our parts.

I did the PCR with the RTTH enzyme, according to the following steps.

1. I prepared the two mixes, which were:

Mix 1

Compound Volume(μL)
H20 9
Buffer rTth[3.3x] 6
Mg(OAc)2[25mM] 3
dNTPs[10mM] 3
Oligo UP[5 pmol/μL] 4
Oligo DW[5 pmol/μL] 4
Diluted DNA (1/50) 1

Mix 2

Compound Volume(μL)
H20 10.5
Buffer rTth 9
rTth DNApol 0.5

2. Once ready a I put the mix into the thermocylcer, the first cycle was:


3. Then I added the mix 2, after that the following cycle were done:

30 cylces:

94°C / 30 seg
60°C / 30 seg
72°C / 3 min

1 cycle:

72°C / 5 min
4° C / ∞

I will see the results on wednesday, in an electrophoresis gel.