Team:UCLA/Notebook/Spider Silk Genetics/28 May 2015
5/28/2015
BsaI Digestion of VF/R Amplified MaSp
- Digested 1.5 ug of each DNA species purified yesterday using 4 uL BsaI in a 50 uL reaction.
| AB (233.2 ng/uL) | BC (301.8 ng/uL) | CA (276.4 ng/uL) | |
|---|---|---|---|
| DNA (1.5 ug) | 6.46 uL | 4.97 uL | 5.43 uL |
| 10x Cutsmart | 5 uL | 5 uL | 5 uL |
| BsaI | 4 uL | 4 uL | 4 uL |
| ddH2O | 34.54 uL | 36.03 uL | 35.57 uL |
| Total | 50 uL | 50 uL | 50 uL |
| 50 C | 2 hrs |
| 65 C | 20 min |
Results
- Cast a 1.8% TAE gel to visualize results. We ran at 140 V for 1 hr 15 min. Used 2 uL of NEB 50 bp ladder.
Fig. 1 VF/R amplified MaSp AB, BC, CA was digested with BsaI as detailed above. The 443 bp band corresponds to undigested fragments. The 184 and 150 bp bands correspond to residual bands from either side of the desired MaSp sequence. The 102 bp band is the desired product with our engineered sticky ends.
- The 443 bp band is rather prominent, it seems that digesting 1.5 ug of VF/R amplified plasmid is too much for BsaI to completely digest.
- Gel extracted the 102 bp bands using Qiagen columns with the following yields:
| Concentration (ng/uL) | A 260/280 | |
|---|---|---|
| AB | 21.47 | 2.51 |
| BC | 51.00 | 3.98 |
| CA | 36.88 | 3.18 |