Team:UCLA/Notebook/Honeybee Silk/8 July 2015
Contents
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 hout |
| Heat Inactivation | 80C | 15 min |
| Hold | 1C | 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