Team:HokkaidoU Japan/Notebook/aggregation Week 11

From 2012.igem.org

(Difference between revisions)
(10th digestion)
(10th digestion recipe)
Line 13: Line 13:
{|class="hokkaidou-table-digestion"
{|class="hokkaidou-table-digestion"
   |-
   |-
-
   |DNA solution ( 30ng/ul)
+
   |DNA solution ( 35ng/ul)
-
   |22 ul
+
   |17 ul
   |-
   |-
-
   |XbaI
+
   |EcoRI
   |1 ul
   |1 ul
   |-
   |-
-
   |PstI
+
   |SpeI
   |1 ul
   |1 ul
   |-
   |-
-
   |10xM buffer
+
   |10xH buffer
   |3 ul
   |3 ul
   |-
   |-
   |DW
   |DW
-
   |3 ul
+
   |8 ul
 +
  |-
 +
  |Total
 +
  |30 ul
 +
  |}
 +
 
 +
 
 +
 
 +
Insert2 (Ag43-dT-pSB1AK3)
 +
{|class="hokkaidou-table-digestion"
 +
  |-
 +
  |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
   |Total
Line 35: Line 62:
-
Vector(pBAD-RBS-pSB1A2)
+
Vector(pSTV28)
{|class="hokkaidou-table-digestion"
{|class="hokkaidou-table-digestion"
   |-
   |-
-
   |DNA solution ( 50ng/ul)
+
   |DNA solution ( 15ng/ul)
-
   |3 ul
+
   |9 ul
   |-
   |-
-
   |SpeI
+
   |EcoRI
   |1 ul
   |1 ul
   |-
   |-
-
   |PstI
+
   |NotI
   |1 ul
   |1 ul
   |-
   |-
   |10xH buffer
   |10xH buffer
   |2 ul
   |2 ul
 +
  |-
 +
  |10xBSA
 +
  |0.2ul
   |-
   |-
   |DW
   |DW
-
   |13 ul
+
   |7 ul
   |-
   |-
   |Total
   |Total
Line 58: Line 88:
-
control (eCFP-pSB1A2)
+
control (pT7-RBS-pSB1C3)
{|class="hokkaidou-table-digestion"
{|class="hokkaidou-table-digestion"
   |-
   |-
-
   |DNA solution (30 ng/ul)
+
   |DNA solution (30~40 ng/ul)
-
   |5 ul
+
   |10 ul
   |-
   |-
   |XbaI
   |XbaI
Line 74: Line 104:
   |-
   |-
   |DW
   |DW
-
   |11 ul
+
   |6 ul
   |-
   |-
   |Total
   |Total
Line 93: Line 123:
|-
|-
|2
|2
-
|60
+
|70
-
|15
+
|20
|-
|-
|3
|3
Line 100: Line 130:
|HOLD
|HOLD
|}
|}
 +
</p>
</div></div>
</div></div>
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi -->
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi -->

Revision as of 05:08, 10 September 2012

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