Team:UESTC-China/Protocol

From 2014.igem.org

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Revision as of 04:44, 24 September 2014

UESTC-China

DNA ligation with T4 DNA Ligase
Component 20ul Reaction
1.33X Master Mix 15ul
Insert DNA the moles of insert DNA to vector DNA is 5:1
>Vector DNA 50ng
ddH2O To 20ul
Incubate at 50℃ for 1 hour
DNA ligation with Gibson Assemble
Component 20ul Reaction
Vector DNA 50ng
Insert DNA the moles of insert DNA to vector DNA is 5:1
10X T4 DNA ligase buffer 2ul
T4 DNA ligase 1ul
ddH2O to 20ul
Incubate at 25℃ for 12 hours
Double Digestion of DNA
Component 50ul Reaction
10x FastDigest buffer 5ul
Restriction enzyme 1 1ul
Restriction enzyme 2 1ul
DNA 1-2ug
ddH2O to 50ul
Incubate at 37℃ for 2 hours
PCR protocol for KOD-Plus-Neo
Component 50ul Reaction
ddH2O 33ul
10X buffer for KOD-Plus-Neo 5ul
MgSO4 1ul
10mM dNTPs 1ug
10uM forward primer 1ul
10uM reverse primer 1ul
Template 1ul
KOD enzyme 1ul