Team:HokkaidoU Japan/Notebook/aggregation Week 11

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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 (previously digested with HindIII) 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


digestion result