Team:UCLA/Notebook/Spider Silk Genetics/26 May 2015
5/26/2015
1. BsaI Digestion for VF/R amplified MaSp2 2CA
- Used all of the DNA from last Friday.
- We set up the digestion according to the following protocol.
| Volume (uL) | |
| DNA (42 ng/uL) | 8.5 |
| DNA (36 ng/uL) | 8.5 |
| 10x Cutsmart | 5 |
| BsaI | 4 |
| ddH2O | 24 |
| Total | 50 |
| 50 C | 2 hrs |
| 65 C | 20 min |
2. Results
- We cast a 2% TAE gel to visualize results. We used 2 uL of NEB 50 bp ladder.
Fig. 1 Results of BsaI digestion of VF/R amplified MaSp2 2CA. Digestion of the fragment is mostly complete (compare to previously). The faint band smear at 443 bp corresponds to undigested fragments. The bands at 150 and 184 correspond to digestion 'leftovers' that contain the VF+prefix and VR+suffix fragments respectively. The band at 100 bp corresponds to our desired digested product containing the sticky ends.
- These results indicate that BsaI digestion of VF/R amplified products are viable, and this could be an alternate route to synthesizing MaSp monomers.
- Future reactions may need to be run for longer periods of time.