Team:UCLA/Notebook/Honeybee Silk/16 July 2015
Contents
Miniprep of 7/15 Cultures
- I miniprepped each of the five samples of Silk in Pet 24a using the Zymo Miniprep kit.
OD:
- 95.98ng/uL
- 108.12ng/uL
- 88.05ng/uL
- 128.87ng/uL
- 99.23ng/uL
Colony PCR of 7/15 Transformation
Transformation Results
Each plate only had one colony present. Both were picked and suspended in 100uL Nuclease Free Water.
Colony PCR Reaction
- using Q5, transformed E. coli, and the VF2 and VR primers in preparation for insertion into psb1c3
- Two reactions, one for each of the colonies.
| Component | Volume (out of 25uL) |
|---|---|
| 5X Q5 Reaction Buffer | 5uL |
| 10mM dNTPS | 0.5uL |
| 10mM VF2 primer | 1.25uL |
| 10mM VR primer | 1.25uL |
| Transformed cells in ddH2O | 1uL |
| Q5 High Fidelity DNA Polymerase | 0.25uL |
| Nuclease Free Water | 15.75uL |
| Step | Temperature | Time |
|---|---|---|
| Initial Denaturation | 98C | 3 min |
| Cycles (x25) | 98C | 10s |
| Annealing | 66C | 15s |
| Extension | 72C | 15s |
| Final Extension | 72C | 2min |
| Hold | 12C | Hold |
- TM calculated using NEB TM Calculator
- Total Run Time 35 minutes (including ramp times)
Gel Visualization
Both samples were run on a 1.5% agarose gel against a 1kb ladder.
Expected band size: ~1600bp
Results:
Re-do of Silk-SpyCatcher Transformation
- There were only one or two very small colonies from our transformation on 7/15.
- We will colony PCR these two colonies
- We will also re-try the ligation of the silk+SpyC into the psb1c3 backbone, this time cleaning up the ligation using zymo clean and concentrator and eluting into 10 ul of H20.
- We can then re try the transformation.