VR-pFC9 Gibson assembly continued #
Continuing Gibson assembly I started yesterday with chemically competent cells.
Transformation results #
The cells seem to still be viable despite being 4 years expired, although they may be extra sensitive to mechanical lysis and/or temperature changes as success rate of transformation was about 50%. The pUC13 control DNA provided as part of the kit was able to transform bacteria confirming their viability independently of Gibson assembly plasmids.
| Insert | Transformation successful? |
|---|---|
| VR-29 | 1 |
| VR-8 | 1 |
| VR-13 | 1 |
| VR-3 | 1 |
| VR-10 | 0 |
| VR-7 | 0 |
| VR-5 | 0 |
| VR-18 | 0 |
I think this may have been due to my handling of the cells. Even though I was careful I did introduce a few bubbles into some samples. Today I will repeat transformation protocol for inserts that failed to transform pFC9 from the Gibson assembly products.
Culture successful transformations #
I extracted single colonies from successful transformations into 500 ml of LB broth with 100 ug/ml of ampicillin. I placed these cultures in the 37C hot room to incubate overnight at 11:45 AM.
Redo failed transformations #
I repeated the protocol included with the chemically competent cells for the transformations that failed (VR 10, 7, 5 and 8) using the extra Gibson assembly products I produced yesterday. I plated these 4 samples and placed into 37C hot room at 1:25 PM to incubate overnight.
Modular 3D printed plate holder is too large #
The modular version of the petri dish holder I printed yesterday is too large for the plates I normally use :(
The dovetails do seem to work well though.
PCR all additional VR inserts #
After plating the reattempted transformations I started a PCR reaction described at this spreadsheet page “8-24-21 Big VR PCR” to amplify all VR inserts that I have not previously amplified.
Nanodrop measurements #
Below is table of nanodrop results of the unpurified PCR products.
| Insert | ng/ul | 26/280 | 260/230 |
|---|---|---|---|
| VR-1 | 94.2 | 0.832 | 0.19 |
| VR-2 | 100.2 | 0.87 | 0.197 |
| VR-4 | 96.2 | 0.854 | 0.195 |
| VR-5 | 90.1 | 0.804 | 0.181 |
| VR-8 | 95 | 0.857 | 0.196 |
| VR-11 | 96.1 | 0.846 | 0.187 |
| VR-12 | 105 | 0.901 | 0.199 |
| VR-14 | 89.1 | 0.836 | 0.189 |
| VR-15 | 91.8 | 0.836 | 0.191 |
| VR-16 | 92.8 | 0.841 | 0.19 |
| VR-17 | 90 | 0.838 | 0.19 |
| VR-19 | 93.1 | 0.915 | 0.192 |
| VR-20 | 90.9 | 0.842 | 0.188 |
| VR-22 | 93.6 | 0.837 | 0.222 |
| VR-23 | 92.1 | 0.847 | 0.191 |
| VR-25 | 96.8 | 0.839 | 0.191 |
VR-30 evaporated or something? It was the last sample I transferred
out from the PCR plate and there was almost nothing in there, maybe
a microliter of sample. I placed all samples into a new box in the
kitchen freezer called VR Inserts 2. All samples added to the box
are shown in the image below.
I am waiting on the Zymogen PCR cleanup spin columns to arrive before I purify and then run all the purified products on gel.