Team:UCLA/Notebook/Honeybee Silk/13 July 2015

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Colony PCR of 7/12 Transformation

• The transformation was successful for the 3:1 ligated products plates. I picked 3 colonies off the plate and suspended each in 100uL of ddH2O.
• Using APEX Taq Red Mastermix

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	30s
Cycles(x30)	95C	10s
Annealing	67C	20s
Extension	70C	30s
Final Extension	70C	2 min
Hold	14C	Hold

Visualization

I ran each of the sample on a 1% agarose gel against a 1kB ladder for 20 minutes. Expected band sizes: ~1000bp

Samples 1 and 3 had bright bands at the appropriate base pair length.

(sorry need to export images as jpeg)

Inoculation

The remaining 99uL of samples 1 and 3 were inoculated with 5mL LB broth and 5uL 1000X kanamycin, incubated at 37C.

Testing competency of BL21 cells from 7/12

• Used puc 19 vector at 50 picograms/ul to test transformation efficiency.
• Added 2 ul of puc 19 to 50 ul of competent BL21 cells
• Used this NEB protocol to transform with our BL21 cells.
• Plated 10% of the cells on a warm amp plate and put plate in at 4 pm 7/13