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

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Double Digestion and Cleanup of pET24a

• Sequencing results checked out for miniprepped pet 24a plasmid.
• I will be using the first sample from 7/9 (OD: 231.26ng/uL)

Digestion Reaction

Component	Volume (out of 50uL)
NEB 2.1 Buffer	5uL
BamHI	1uL
NdeI	1uL
pET24a (Miniprepped)	5uL
Nuclease Free Water	38uL

Program

Step	Temperature	Time
Incubation	37C	1 hour
Heat Inactivation	80C	15 min
Hold	10C	Hold

I ran the sample on a 1% agarose gel against a 1kB ladder. The appropriate band length was present (5310 bp).

(picture, do this asap)

The band was excised from the gel and purified along with the collected gel sample of the honeybee insert from 7/8 using the Qiagen Gel Purification kit.

OD: Honeybee silk: 28.4ng/uL pET24a: 23.6

Ligation

• 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