Team:UCLA/Notebook/Spider Silk Genetics/29 May 2015
5/29/2015
Ligation Test using VF/R amplified Plasmids
- Ligation using T7 Ligase. 50 ng each DNA in 15 uL reactions. The DNA was taken from yesterday's experiments.
- We used the following protocol:
| Concentration (ng/uL) | A 260/280 | |
|---|---|---|
| AB | 21.47 | 2.51 |
| BC | 51.00 | 3.98 |
| CA | 36.88 | 3.18 |
| AB+BC | BC+CA | CA+AB | |
|---|---|---|---|
| 2x T7 Ligase Buffer | 7.5 uL | 7.5 uL | 7.5 uL |
| DNA 1 | 2.33 uL | 1 uL | 1.36 |
| DNA 2 | 1 uL | 1.36 | 2.33 uL |
| T7 Ligase | 0.5 uL | 0.5 uL | 0.5 uL |
| ddH2O | 3.67 uL | 4.64 uL | 3.31 uL |
| Total | 15 uL | 15 uL | 15 uL |
- Reactions were incubated at 25 C for 10 minutes, then immediately run on gel.
Results
- Cast 2% TAE agarose gel, used 2 uL NEB 50 bp ladder.
Fig. 1 T7 Ligation test using VF/R amplified MaSp. Lane 1 is AB+BC, Lane 2 is BC+CA, Lane 3 is CA+AB. 204 bp band corresponds ligated product. The 102 bp band corresponds to unligated fragments. Compare to previous results from 5/12/2015 derived from plasmid.
- These results indicate that both plasmid and PCR product digestion can be used for ICA work. Our next steps are to characterize other parts used in ICA.
Others
- Ordered Initiator, Terminator and Caps with new sticky ends