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 ( 35ng/ul) | 17 ul |
EcoRI | 1 ul |
SpeI | 1 ul |
10xH buffer | 3 ul |
DW | 8 ul |
Total | 30 ul |
Insert2 (Ag43-dT-pSB1AK3)
DNA solution ( 35ng/ul) | 25 ul |
XbaI | 1 ul |
NotI | 1 ul |
10xK buffer | 1.5 ul |
100xBSA | 0.3ul |
DW | 1.5 ul |
Total | 30 ul |
Vector(pSTV28)
DNA solution ( 15ng/ul) | 9 ul |
EcoRI | 1 ul |
NotI | 1 ul |
10xH buffer | 2 ul |
10xBSA | 0.2ul |
DW | 7 ul |
Total | 20 ul |
control (pT7-RBS-pSB1C3)
DNA solution (30~40 ng/ul) | 10 ul |
XbaI | 1 ul |
SpeI | 1 ul |
10xM buffer | 2 ul |
DW | 6 ul |
Total | 20 ul |
Number | Degree | Minute |
1 | 37 | 120 |
2 | 70 | 20 |
3 | 4 | HOLD |