"
Team:HokkaidoU Japan/Notebook/aggregation Week 11
From 2012.igem.org
September 10th
Digestion of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2
To make a construct of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 by 3piece ligation, we digested pBAD-RBS-eCFP-RBS-pSB1A2 with EcoRI & SpeI, Ag43-dT-pSB1AK3 with XbaI & NotI and pSTV28 with EcoRI and NotI. Then we digested pT7-RBS-pSB1C3 with XbaI & SpeI as a control for confirmation of the ability of restriction enzyme. Insert1 (pBAD-RBS-eCFP-RBS-pSB1A2)
| DNA solution ( 30ng/ul) | 22 ul |
| XbaI | 1 ul |
| PstI | 1 ul |
| 10xM buffer | 3 ul |
| DW | 3 ul |
| Total | 30 ul |
Vector(pBAD-RBS-pSB1A2)
| DNA solution ( 50ng/ul) | 3 ul |
| SpeI | 1 ul |
| PstI | 1 ul |
| 10xH buffer | 2 ul |
| DW | 13 ul |
| Total | 20 ul |
control (eCFP-pSB1A2)
| DNA solution (30 ng/ul) | 5 ul |
| XbaI | 1 ul |
| SpeI | 1 ul |
| 10xM buffer | 2 ul |
| DW | 11 ul |
| Total | 20 ul |
| Number | Degree | Minute |
| 1 | 37 | 120 |
| 2 | 60 | 15 |
| 3 | 4 | HOLD |
</div></div>
</div>
</div>
</div>
</div>
