Team:UCLA/Notebook/Honeybee Silk/19 August 2015

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Colony PCR of 8/18 Transformation

8 colonies were picked off the plates and suspended in 100uL ddH2O.

Component	Volume (out of 25uL)
APEX Taq Red Mastermix	12.5uL
10mM primer 10	1.25uL
10mM primer 11	1.25uL
Transformed Cells	1uL
Nuclease Free Water	9uL

Program

Step	Temperature	Time
Initial Denaturation	95C	3 min
Cycles(x30)	95C	15s
Annealing	67C	30s
Extension	70C	2 min
Final Extension	70C	2 min
Hold	14C	Hold

The samples were run on a 1% agarose gel against 1kB ladder. Expected band size: ~2100bp

Results:

[insert gel image here]

Samples 1, 4, and 8 had the cleanest bands, these three samples are going to be inoculated.

Inoculation of Sample 1, 4, and 8

The remaining 99uL of the suspended cells were each inoculated in 5mL LB broth with 5uL 1000X chloramphenicol.