Team:HokkaidoU Japan/Notebook/aggregation Week 10

From 2012.igem.org

(Difference between revisions)
(3e)
(3rd digestion-transformation)
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#Incubated at 37C for  hrs.
#Incubated at 37C for  hrs.
</p>
</p>
 +
 +
 +
==Estimation of concentration of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2==
 +
<p>
 +
No.2,5 means the colony number of colony PCR of eCFP-RBS-pSB1A2.
 +
 +
 +
[[image:|thumb|electrophoresis result]]
 +
 +
 +
We estimated the concentration of eCFP-RBS-pSB1A2 is 30 ng/ul  and pBAD-RBS-pSB1A2 is 50ng/ul.
 +
</p>
 +
 +
==Digestion of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2==
==Digestion of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2==
Line 84: Line 98:
{|class="hokkaidou-table-digestion"
{|class="hokkaidou-table-digestion"
   |-
   |-
-
   |DNA solution ( 35ng/ul)
+
   |DNA solution ( 30ng/ul)
-
   |11 ul
+
   |22 ul
   |-
   |-
-
   |EcoRI
+
   |XbaI
   |1 ul
   |1 ul
   |-
   |-
-
   |SpeI
+
   |PstI
   |1 ul
   |1 ul
   |-
   |-
-
   |10xH buffer
+
   |10xM buffer
-
   |2 ul
+
   |3 ul
   |-
   |-
   |DW
   |DW
-
   |5 ul
+
   |3 ul
   |-
   |-
   |Total
   |Total
-
   |20 ul
+
   |30 ul
   |}
   |}
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{|class="hokkaidou-table-digestion"
{|class="hokkaidou-table-digestion"
   |-
   |-
-
   |DNA solution ( 29ng/ul)
+
   |DNA solution ( 50ng/ul)
-
   |6 ul
+
   |3 ul
   |-
   |-
-
   |EcoRI
+
   |SpeI
   |1 ul
   |1 ul
   |-
   |-
-
   |XbaI
+
   |PstI
   |1 ul
   |1 ul
   |-
   |-
-
   |10xM buffer
+
   |10xH buffer
   |2 ul
   |2 ul
   |-
   |-
   |DW
   |DW
-
   |10 ul
+
   |13 ul
   |-
   |-
   |Total
   |Total
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-
control (pT7-RBS-pSB1C3)
+
control (eCFP-pSB1A2)
{|class="hokkaidou-table-digestion"
{|class="hokkaidou-table-digestion"
   |-
   |-
   |DNA solution (30 ng/ul)
   |DNA solution (30 ng/ul)
-
   |6 ul
+
   |5 ul
   |-
   |-
   |XbaI
   |XbaI
 +
  |1 ul
 +
  |-
 +
  |SpeI
   |1 ul
   |1 ul
   |-
   |-
   |10xM buffer
   |10xM buffer
   |2 ul
   |2 ul
-
  |-
 
-
  |100x BSA
 
-
  |0.2
 
   |-
   |-
   |DW
   |DW
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-
[[image:HokkaidoU2012 120902 Digestion I-+ V-+ C(XbaI)-+.jpg|thumb|digestion result]]
+
[[image:|thumb|digestion result]]
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</p>
</p>
-
==Estimation of concentration of eCFP, pT7-RBS-Ag43-dT-pSB1C3 and Ag43-dT-pSB1AK3==
 
-
<p>
 
-
No.2,5 means the colony number of colony PCR of eCFP-RBS-pSB1A2.
 
-
 
+
==Ethanol precipitation of digestion products (eCFP-RBS and pBAD-RBS-pSB1A2) and estimation of concentration==
-
[[image:.jpg|thumb|electrophoresis result]]
+
-
 
+
-
 
+
-
We estimated the concentration of pT7-RBS-Ag43-dT-pSB1C3 is 31 ng/ul  and Ag43-dT-pSB1AK3 is 35ng/ul.
+
-
</p>
+
-
 
+
-
==Ethanol precipitation of digestion products (eCFP and RBS-pSB1A2) and estimation of concentration==
+
<p>
<p>
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
-
#Centrifuged in 14000 rpm, 30 min at 4C.
 
-
#Remove supernatant and added 220 ul of 70% ethanol.
 
#Centrifuged in 15000 rpm, 15 min at 4C.
#Centrifuged in 15000 rpm, 15 min at 4C.
 +
#Remove supernatant and added 220 ul of 70% ethanol.
 +
#Centrifuged in 15000 rpm, 10 min at 4C.
#Remove supernatant and air drying in room temperature then added 5 ul of DW.  
#Remove supernatant and air drying in room temperature then added 5 ul of DW.  
-
[[image:HokkaidoU2012 120902 Ethapre V I.jpg|thumb|ethanol precipitation result]]
+
[[image:|thumb|ethanol precipitation result]]
-
We estimated the concentration of ethanol presipitation products.The concentration of Insert DNA solution is about 20 ng/ul and Vector DNA solution is about 37 ng/ul.
+
We estimated the concentration of ethanol presipitation products.The concentration of Insert DNA solution is about 20 ng/ul and Vector DNA solution is about 50 ng/ul.
</p>
</p>
-
==Ligation of eCFP and RBS-pSB1A2==
+
==Ligation of eCFP-RBS and pBAD-RBS-pSB1A2==
<p>
<p>
{|class="hokkaidou-table-ligation"
{|class="hokkaidou-table-ligation"
|-
|-
-
|Vector DNA (37 ng/ul)
+
|Vector DNA (50 ng/ul)
-
|1 ul
+
|1.5 ul
|-
|-
-
|Insert DNA (20 ng/ul
+
|Insert DNA (20 ng/ul)
|4 ul
|4 ul
|-
|-
|Ligation Mighty Mix
|Ligation Mighty Mix
|6 ul
|6 ul
 +
|-
 +
|DW
 +
|0.5 ul
|-
|-
|Total
|Total
-
|11 ul
+
|12 ul
|}
|}
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</p>
</p>
-
==Transformation of eCFP-RBS-pSB1A2==
+
==Transformation of pBAD-RBS-eCFP-RBS-pSB1A2==
<p>
<p>
-
#Mixed 2 ul eCFP-RBS-pSB1A2 ligation product to 50 ul of thawed competent cells on ice.
+
#Mixed 2 ul pBAD-RBS-eCFP-RBS-pSB1A2 ligation product to 50 ul of thawed competent cells on ice.
#Incubated on ice for 30 min.
#Incubated on ice for 30 min.
#Mixed 350 ul of LB.   
#Mixed 350 ul of LB.   
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#Plated 300 ul of the culture onto first dish and spread.
#Plated 300 ul of the culture onto first dish and spread.
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.
-
#Incubated the plates at 37C for 19 hours.
+
#Incubated the plates at 37C for hours.
</p>
</p>

Revision as of 21:40, 3 September 2012

Contents

September 3rd

Colony PCR of eCFP-RBS-pSB1A2

DNA solution 4 ul
Kapa-Taq(Taq polymerase) 5 ul
Forward Primer(EX-F primer) 0.5 ul
Reverse Primer(PS-R down primer) 0.5 ul
Total 10 ul


Number Degree Second
1 95 120
2 95 30
3 68.9 30
4 72 60
5 72 60
6 4 HOLD

Cycle:2~4 x 35

We used N1 (DW only) and N2 (ptetR-RBS-eCFP-dT-pSB1A2) as controls. Desired product is about 776bp.

Colony PCR result


We confirmed that about 70% of ligated DNA formed our desired construct. We selected No.2 and 5 colony for incubation and store No.7 and 8 colony mixture at 4C.

Incubation of eCFP-RBS-pSB1A2 for mini-prep

  1. Prepared 2 ml LBA into culture tubes.
  2. Re-suspended 2 colony mixture (No.2 and No.5 respectively).
  3. Incubated at 37C for hrs.


Estimation of concentration of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2

No.2,5 means the colony number of colony PCR of eCFP-RBS-pSB1A2. [[image:|thumb|electrophoresis result]] We estimated the concentration of eCFP-RBS-pSB1A2 is 30 ng/ul and pBAD-RBS-pSB1A2 is 50ng/ul.


Digestion of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2

To make a construct of pBAD-RBS-eCFP-RBS-pSB1A2, we digested eCFP-RBS-pSB1A2 with XbaI & PstI and pBAD-RBS-pSB1A2 with SpeI & PstI. And we digested eCFP-pSB1A2 with XbaI & SpeI as a control for confirmation of the ability to digest. Insert (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


[[image:|thumb|digestion result]]


From this image, we confirmed that DNA were digested into fragments and all of restriction enzyme worked.


Ethanol precipitation of digestion products (eCFP-RBS and pBAD-RBS-pSB1A2) and estimation of concentration

  1. Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
  2. Centrifuged in 15000 rpm, 15 min at 4C.
  3. Remove supernatant and added 220 ul of 70% ethanol.
  4. Centrifuged in 15000 rpm, 10 min at 4C.
  5. Remove supernatant and air drying in room temperature then added 5 ul of DW.
[[image:|thumb|ethanol precipitation result]] We estimated the concentration of ethanol presipitation products.The concentration of Insert DNA solution is about 20 ng/ul and Vector DNA solution is about 50 ng/ul.

Ligation of eCFP-RBS and pBAD-RBS-pSB1A2

Vector DNA (50 ng/ul) 1.5 ul
Insert DNA (20 ng/ul) 4 ul
Ligation Mighty Mix 6 ul
DW 0.5 ul
Total 12 ul


Ligation reaction time was in detail below.

Degree Minute
16 30
65 10
4 Hold

Transformation of pBAD-RBS-eCFP-RBS-pSB1A2

  1. Mixed 2 ul pBAD-RBS-eCFP-RBS-pSB1A2 ligation product to 50 ul of thawed competent cells on ice.
  2. Incubated on ice for 30 min.
  3. Mixed 350 ul of LB.
  4. Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBA).
  5. Plated 300 ul of the culture onto first dish and spread.
  6. Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.
  7. Incubated the plates at 37C for hours.



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