Team:UCLA/Notebook/Spider Silk Genetics/11 May 2015
5/11/2015
1 2AB, 2BC, 2CA Ligation Test
1.1 BsaI digestion of 2AB, 2BC, 2CA
Used 5 ug of each DNA.
| 2AB (112 ng/uL) | 2BC (205 ng/uL) | 2CA (218.7 ng/uL) | |
| BsaI | 4 uL | 4 uL | 4 uL |
| Template | 44.6 uL | 24.39 uL | 22.86 uL |
| Cutsmart | 5 uL | 5 uL | 5 uL |
| ddH2O | 0 uL | 16.61 uL | 18.14 uL |
| Total | 53.6 uL | 50 uL | 50 uL |
| 50 C | 2 hr | ||
| 65 C | 20 min |
We are conducting ligation tests between all possible combinations using T4 and T7 ligase
2 NotI Concatemers
Sri suggested using NotI to digest the plasmids and create a super plasmid that
contains multiple copies of the monomers.
Testing NotI digestion today using 2CA with the protocol below.
| 2CA (280 ng/uL) | |
| NotI-HF | 2 uL |
| DNA | 7.2 uL |
| Cutsmart | 5 uL |
| ddH2O | 35.8 uL |
| Total | 50 uL |
| 37 C | 1 hr |
| 65 C | 20 min |
3 Results
Cast 200 mL of a 1.5% TAE agarose gel to visualize results of both BsaI and Not1 digestion.
Figure 1: Digestion Results: The 100 bp bands were excised as well as the
second band in the NotI digestion.