Team:UCLA/Notebook/Honeybee Silk/30 May 2015

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Gel Visualization of 5/26 PCR

The PCR products from 5/26 were run on a 1% agarose gel against a 1kB ladder. I used 5 uL of each sample and added 1uL of loading dye.

Gel Image

Results:

There were bands present for the correct base pair length (~1000bp). The sample run at 67C for the annealing step produced the cleanest, brightest band.

PCR of Honeybee G-block (Optimal)

From the gel visualization, 67C annealing temperature produced the best results. I am running a 50uL PCR reaction now under the optimal conditions.

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

Purification of PCR product

I purified my PCR product using Qiagen MinElute columns, following their protocol.

OD: 349.18ng/uL