Team:UCLA/Notebook/Honeybee Silk/10 July 2015

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Cloning Honeybee Silk into Pet 24a

• Sequencing results checked out for miniprepped pet 24a plasmid.
• Double digested PET 24a with BamH1 and Nde1 restriction enzymes
• Ran on gel, excised, and purified
• Ran ligation reaction with double digested pet 24a and honeybee sequenced

Component 3:1 insert:vector)	Volume (out of 20uL)
T4 Ligase Buffer	2 ul
Pet 24a backbone (23.6 ng/ul)	2.54 ul
SIlk insert 28.4 ng/ul	1.12
T4 Ligase	1 ul
Water	13.34

Component 5:1 insert to vector	Volume (out of 20uL)
T4 Ligase Buffer	2 ul
Pet 24a backbone (23.6 ng/ul)	2.54 ul
SIlk insert 28.4 ng/ul	1.9
T4 Ligase	1 ul
Water	12.56

Running ligation reaction overnight 16 C starting at 6:45 pm 7/10