Difference between revisions of "Team:UCLA/Notebook/Honeybee Silk/8 July 2015"

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Revision as of 04:07, 11 July 2015

Inoculation of 5/19 Transformation

Recent experiments have proven that my pET24a miniprepped sample was not correct. Today I will repick colonies and grow them up for miniprepping.

I picked two colonies from the 1:1 kanamycin plate and suspended them in 100uL ddH2O. Each sample was incubated at 37C with 5mL LB broth and 5uL 1000X kanamycin.


PCR of Honeybee G-block and Purification

The concentration obtained after digestion and purification was too low (20.12ng/uL), today I will be redoing the PCR, digestion, and purification to obtain a higher yield.

Component Volume (out of 50uL)
5X Q5 Reaction Buffer 10uL
10mM dNTPS 1uL
10mM primer 10 2.5uL
10mM primer 11 2.5uL
Template (Honeybee G-block) 1uL
Q5 High Fidelity DNA Polymerase 0.5uL
Nuclease Free Water 32.5uL

Program

Step Temperature Time
Initial Denaturation 98C 30s
Cycles (x25) 98C 10s
Annealing 67C 20s
Extension 72C 30s
Final Extension 72C 2min
Hold 12C Hold

The sample was then purified using the Zymo DNA Concentrate and Cleanup Kit. OD: 283.09ng/uL

Digestion and Purification

Component Volume (out of 50uL)
NEB 2.1 Buffer 5uL
BamHI 1uL
NdeI 1uL
PCR Purified Honeybee G-block 5uL
Nuclease Free Water 38uL

Program

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


The sample was loaded onto a 1% agarose gel with 10uL 6X loading dye against a 1kB ladder. Expected band size: ~1000bp Results:


The digested sample was purified using the Qiagen Gel