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From 2010.igem.org

Module I, group 2c

Since we have got no positive results for mutiple-fragment ligation, we really want to know the efficiency of it. Therefore, we use the ligation product as template, upstream primer of eGFP and downstream primer of Kan to run PCR, after which gel is run to check whether there is any ligation product of three fragments-eGFP, mCherry and Kan.

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PCR system

ligation product       1ul
up-primer              1ul
down-primer            1ul
H2O                    7ul
Supermix               10ul

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Result: ligation product of three fragments is little.