Team:Hannover/Protocols/Cloning/EMP
From 2014.igem.org
Protocols / Exponential Megaprimer PCR (EMP)
The exponential megaprimer PCR (EMP) was used to replace the metal-binding-related sequences of our pORE-E3_2x35S_T4MBP by GFP. In theory, the EMP is based on two PCR reaction: while the insert is amplified by a standard PCR in the first (megaprimer), the second integrates the places the insert in the final vector backbone already. To try different weight ratios of vector backbone and megaprimer turned out be helpful in prior experiments. Since a correct nucleotide sequence was important here, the F-530 Phusion® High-Fidelity DNA Polymerase (Thermo Scientific) was used.
Table 1: EMP Mixes
Test different ratios of insert and vector DNA amounts. We recommend fours mixes: two containing 10 and 20 ng of insert respectively and two containing 80 and 160 ng of insert but no F1 oligonucleotide.
EMP temperature program
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