Team:UCLA/Notebook/Spider Silk Genetics/20 April 2015
4/20/2015
1 PCR Amplification of pSB1C3
∙ Due to poor amplification of pSB1C3 from friday, we are doing a temperature test today.
∙ Followed same protocol as 4/17/2015, but scaled to 3x 25 uL reactions.
∙ Decided to take nanodrop reading of the template Julian provided. It was 230 ng/uL. Instead of using stock concentration of template, we will use a 1:100 dilution. Results shown below.
∙ We gel extracted the bands together. 26.4 ng/uL, A 260/280: 1.56.
2 EcoRI and PstI Digestion of MaSp 2AB, 2BC, 2CA, and pSB1C3
∙ We pooled together all the linearized pSB1C3 that we had. Final concentration: 16.5 ng/uL.
| Concentration (ng/uL) | |
| 2AB | 37.58 |
| 2BC | 42.8 |
| 2CA | 79.11 |
| pSB1C3 | 16.5 |
| 2AB | 2BC | 2CA | pSB1C3 | |
| EcoRI | 1 uL | 1 uL | 1 uL | 1 uL |
| PstI | 1 uL | 1 uL | 1 uL | 1uL |
| 10x NEbuffer 2.1 | 5 uL | 5 uL | 5 uL | 5 uL |
| DNA (500 ng) | 16 uL | 11 uL | 6 uL | 19 uL |
| ddH2O | 27 uL | 32 uL | 37 uL | 24 uL |
| Total | 50 uL | 50 uL | 50 uL | 50 uL |
| 37 C | 1 hr |
| 65 C | 20 min |
| 12 C | hold |
∙ Column purified all DNA after digestion using Zymo kit. Eluted in 10 uL of EB. Results shown below:
| Concentration (ng/uL) | A 260/280 | |
| 2AB | 43.28 | 1.9 |
| 2BC | 34.56 | 1.7 |
| 2CA | 34.9 | 1.7 |
| pSB1C3 | 33.7 | 1.61 |