IGEM:UNAM-Genomics Mexico/2009/Notebook/H2 Pae/2011/08/31

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Abstract

  • Repeating the standardization

Main

Standardization

Today I did a PCR amplifying the RFP flanked by the standard region, this was done with the correct forward primer. To each primer was added a restriction site, sacI and apaI respectively in order to do a ligation by sticky ends with a digested pBBR1MCS-5 plasmid.

The PCR reaction was done according to the following:

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:

94°C/6min

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

30 cylces:

94°C / 30 seg
65°C / 30 seg
72°C / 1.5 min

1 cycle:

72°C / 5 min
4° C / ∞