Team:UCLA/Notebook/Spider Silk Genetics/12 May 2015
5/12/2015
1 Gel Extraction
Performed gel extraction (Qiagen kit) of bands excised from yesterday’s gel. Results below:
| Concentration (ng/uL) | A 260/280 | |
| 2AB | 41.5 | 4.47 |
| 2BC | 54.3 | 3.96 |
| 2CA | 17.6 | 3.21 |
| 2CA NotI ins. | 52.63 | 3.70 |
| 2CA NotI vec. | 112.12 | 2.50 |
2 2AB, 2BC, 2CA BsaI Ligation Test
Set up ligation tests for remaining conditions (see entry for 5/8/2015)for other tests. For each reaction, we used 50 ng of each DNA species.
2.1 T4 Ligase Reactions
| 2AB + 2BC | 2BC +2CA | |
| 10X T4 Ligase Buffer | 1.5 uL | 1.5 uL |
| 2AB (6.82 ng/uL) | 7.3 uL | 0 uL |
| 2BC (54.3 ng/uL) | 1 uL | 1 uL |
| 2CA (17.6 ng/uL) | 0 uL | 2.84 uL |
| T4 Ligase | 1 uL | 1 uL |
| ddH2O | 4.2 uL | 8.66 uL |
| Total | 15 uL | 15 uL |
Reactions were incubated at 25 C for 10 min.
2.2 T7 Ligase Reactions
| 2CA + 2AB | 2BC + 2CA | |
| 2X T7 Ligase Buffer | 7.5 uL | 7.5 uL |
| 2AB | 1.2 uL | 0 uL |
| 2BC | 0 uL | 1 uL |
| 2CA | 2.84 uL | 2.84 uL |
| T7 Ligase | 0.5 uL | 0.5 uL |
| ddH2O | 2.96 uL | 3.16 uL |
| Total | 15 uL | 15 uL |
Reactions were incubated at 25 C for 10 min.
3 Results
We cast a 2% TAE agarose gel to visualize the results. We used 2 uL of 50 bp ladder. See below.
