Team:HokkaidoU Japan/Notebook/aggregation Week 12

From 2012.igem.org

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(19th)
 
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<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
==September 17th==
+
===September 17th===
-
<div>
+
<div class="hokkaidou-section">
-
==Digestion of RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 and ptet-pSB1A2==
+
====Digestion of RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 and ptet-pSB1A2====
-
<p>
+
To make a construct of ptet-RBS-phaC-RBS-phaA-RBS-phaB-RBS-dT-pSB1A2, we digested RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 with XbaI and PstI, ptet-pSB1A2 with SpeI and PstI.
-
To make a construct of ptet-RBS-phaC-RBS-phaA-RBS-phaB-RBS-dT-pSB1A2, we digested RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 with XbaI & PstI, ptet-pSB1A2 with SpeI & PstI.
+
<br />
-
 
+
Insert (RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2)
Insert (RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2)
{|class="hokkaidou-table-digestion"
{|class="hokkaidou-table-digestion"
   |-
   |-
-
   |DNA solution ( 45=50ng/ul)
+
   |DNA solution (45~50 ng/ul)
   |27 ul
   |27 ul
   |-
   |-
Line 96: Line 95:
-
[[image:|thumb|digestion result]]
+
====Ethanol precipitation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2====
-
 
+
-
</p>
+
-
 
+
-
==Ethanol precipitation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2==
+
-
<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 15000 rpm, 15 min at 4C.
+
#Centrifuged at 15000 rpm, 15 min at 4C.
-
#Remove supernatant and added 220 ul of 70% ethanol.
+
#Removed supernatant and added 220 ul of 70% ethanol.
-
#Centrifuged in 15000 rpm, 10 min at 4C.
+
#Centrifuged at 15000 rpm, 10 min at 4C.
-
#Remove supernatant and air drying in room temperature then added 5 ul of DW.  
+
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW.  
-
 
+
-
[[image:|thumb|ethanol precipitation result]]
 
We confirmed that the concentration of Insert DNA solution is 60~70 ng/ul and Vector DNA solution is about 20~30 ng/ul.
We confirmed that the concentration of Insert DNA solution is 60~70 ng/ul and Vector DNA solution is about 20~30 ng/ul.
-
</p>
 
-
==Ligation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2==
+
====Ligation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2====
-
<p>
+
{|class="hokkaidou-table-ligation"
{|class="hokkaidou-table-ligation"
|-
|-
Line 148: Line 138:
|}
|}
-
</p>
+
====Transformation of RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2====
-
 
+
Transformation of ligation ligation product into JM109.
-
==Transformation of RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2==
+
-
<p>
+
-
Transformation of JM109.
+
#Mixed 2 ul RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 ligation product to 50 ul of thawed competent cells on ice.
#Mixed 2 ul RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 ligation product to 50 ul of thawed competent cells on ice.
#Incubated on ice for 30 min.
#Incubated on ice for 30 min.
Line 159: Line 146:
#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 17 hours.
+
#Incubated the plates at 37C for 17 hrs.
-
</p>
+
 
 +
<br style="line-height: 0; clear: both;" />
 +
 
</div></div>
</div></div>
-
 
<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
==September 18th==
+
===September 18th===
-
<div>
+
<div class="hokkaidou-section">
-
==Colony PCR of pBAD-RBS-eCFP-RBS-Ag43-dI-pSB1C3==
+
====Colony PCR of pBAD-RBS-eCFP-RBS-Ag43-dI-pSB1C3====
-
<p>
+
We did colony PCR two times.
We did colony PCR two times.
-
 
{|class="hokkaidou-table-pcr-reagent"
{|class="hokkaidou-table-pcr-reagent"
|-
|-
Line 225: Line 211:
|}
|}
Cycle:2~4 x 35
Cycle:2~4 x 35
-
+
 
We used N1 (DW only) and N2(pBAD-RBS-Ag43-dT-pSB1A2)as controls.
We used N1 (DW only) and N2(pBAD-RBS-Ag43-dT-pSB1A2)as controls.
Desired product is about 702bp.
Desired product is about 702bp.
-
[[image:|thumb|Colony PCR result]]
+
[[image:HokkaidoU2012 120918 colop pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3.jpg|thumb|Colony PCR result]]
-
 
+
[[image:HokkaidoU2012 120918 colop2 pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3.jpg|thumb|Colony PCR result 2]]
-
 
+
-
Target products didn't exist in all samples. We noticed the reason why such results shown is that we used incorrect pSB1C3 DNA solution which isn't confirmed the sequence. We'll try the synthesis once again in today. 
+
-
</p>
+
-
==Digestion of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 and pSB1C3==
+
Target products didn't exist in all samples. We noticed the reason why such results shown is that we used incorrect pSB1C3 DNA solution which isn't confirmed the sequence. We'll try the synthesis once more.
-
<p>
+
-
To make a construct of pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3, we digested pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 with EcoRI & SpeI and pSB1C3 with EcoRI & SpeI.
+
 +
====Digestion of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 and pSB1C3====
 +
To make a construct of pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3, we digested pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 with EcoRI and SpeI and pSB1C3 with EcoRI and SpeI.
 +
<br />
Insert (pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28)
Insert (pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28)
{|class="hokkaidou-table-digestion"
{|class="hokkaidou-table-digestion"
Line 303: Line 287:
-
[[image:|thumb|digestion result]]
+
[[image:HokkaidoU2012 120916 Digestion Insert Vector Control.jpg |thumb|digestion result]]
-
</p>
+
====Ethanol precipitation of pBAD-RBS-eCFP-RBS-Ag43-dT--pSB1C3====
-
==Ethanol precipitation of pBAD-RBS-eCFP-RBS-Ag43-dT--pSB1C3==
+
-
<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 15000 rpm, 15 min at 4C.
+
#Centrifuged at 15000 rpm, 15 min at 4C.
-
#Remove supernatant and added 220 ul of 70% ethanol.
+
#Removed supernatant and added 220 ul of 70% ethanol.
-
#Centrifuged in 15000 rpm, 10 min at 4C.
+
#Centrifuged at 15000 rpm, 10 min at 4C.
-
#Remove supernatant and air drying in room temperature then added 5 ul of DW.  
+
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW.  
 +
[[image:HokkaidoU2012 120916 Ethapre Insert Vector.jpg|thumb|ethanol precipitation result]]
-
[[image:|thumb|ethanol precipitation result]]
+
====Ligation of pBAD-RBS-eCFP-RBS-Ag43-dT and pSB1C3====
-
 
+
-
</p>
+
-
 
+
-
==Ligation of pBAD-RBS-eCFP-RBS-Ag43-dT and pSB1C3==
+
-
<p>
+
{|class="hokkaidou-table-ligation"
{|class="hokkaidou-table-ligation"
|-
|-
Line 354: Line 332:
|}
|}
-
</p>
+
====Transformation of pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3====
-
 
+
Transformation ligation product into JM109.
-
==Transformation of pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3==
+
-
<p>
+
-
Transformation of JM109.
+
#Mixed 2 ul pBAD-RBS-eCFP-RBS-Ag43-dT--pSB1C3 ligation product to 50 ul of thawed competent cells on ice.
#Mixed 2 ul pBAD-RBS-eCFP-RBS-Ag43-dT--pSB1C3 ligation product to 50 ul of thawed competent cells on ice.
#Incubated on ice for 30 min.
#Incubated on ice for 30 min.
Line 366: Line 341:
#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 15 hours.
+
#Incubated the plates at 37C for 15 hrs.
-
</p>
+
 
 +
<br style="line-height: 0; clear: both;" />
</div></div>
</div></div>
 +
<div class="hokkaidou-notebook-daily">
 +
===September 19th===
 +
<div class="hokkaidou-section">
 +
====Colony PCR of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28====
 +
Colony PCR to confirm whether pSTV28 vettor has pBAS-RBs-eCFP-RBs-Ag43-dT as insert or not.
 +
{|class="hokkaidou-table-pcr-reagent"
 +
|-
 +
|DNA solution
 +
|4 ul
 +
|-
 +
|Kapa-Taq(Taq polymerase)
 +
|10 ul
 +
|-
 +
|Forward Primer(200bp down primer)
 +
|0.8 ul
 +
|-
 +
|Reverse Primer(ag43-f4 primer)
 +
|0.8 ul
 +
|-
 +
|DW
 +
|4.4 ul
 +
|-
 +
|Total
 +
|20 ul
 +
|}
 +
 +
 +
{|class="hokkaidou-table-pcr-time"
 +
|-
 +
|Number
 +
|Degree
 +
|Second
 +
|-
 +
|1
 +
|95
 +
|120
 +
|-
 +
|2
 +
|95
 +
|30
 +
|-
 +
|3
 +
|53.3
 +
|30
 +
|-
 +
|4
 +
|72
 +
|180
 +
|-
 +
|5
 +
|72
 +
|60
 +
|-
 +
|6
 +
|4
 +
|HOLD
 +
|}
 +
Cycle:2~4 x 35
 +
 +
 +
We used N1 (DW only) and N2(pBAD-RBS-Ag43-dT-pSB1A2)as controls.
 +
Desired product is about 702bp.
 +
 +
[[image:HokkaidoU2012 120919 colop2 pBAD-RBS-eCFP-RBS-AG43-dT-pSTV28(pSB1C3).jpg|thumb|Colony PCR result]]
 +
 +
We noticed the some of colones have pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 construct as plasmid DNA.
 +
 +
====Colony PCR of pBAD-RBS-eCFP-RBS-Ag43-dI-pSB1C3 and pBAD-RBS-eCFP-RBS-Ag43-dI on pSB1C3====
 +
 +
Colony PCR to confirm whether the constructs written above really have eCFP and Ag43 coding sites or not by using primers: one can anneal to a part of eCFP coding site and another can anneal to a part of Ag43 coding site.
 +
{|class="hokkaidou-table-pcr-reagent"
 +
|-
 +
|DNA solution
 +
|4 ul
 +
|-
 +
|Kapa-Taq(Taq polymerase)
 +
|10 ul
 +
|-
 +
|Forward Primer(FP-F primer)
 +
|0.8 ul
 +
|-
 +
|Reverse Primer(ag43-R primer)
 +
|0.8 ul
 +
|-
 +
|DW
 +
|4.4 ul
 +
|-
 +
|Total
 +
|20 ul
 +
|}
 +
 +
 +
{|class="hokkaidou-table-pcr-time"
 +
|-
 +
|Number
 +
|Degree
 +
|Second
 +
|-
 +
|1
 +
|95
 +
|120
 +
|-
 +
|2
 +
|95
 +
|30
 +
|-
 +
|3
 +
|53.3
 +
|30
 +
|-
 +
|4
 +
|72
 +
|180
 +
|-
 +
|5
 +
|72
 +
|60
 +
|-
 +
|6
 +
|4
 +
|HOLD
 +
|}
 +
Cycle:2~4 x 35
 +
 +
We used N1, N2 (DW only) as controls.
 +
Desired product is about 452 bp and 697 bp respectively.
 +
 +
[[image:HokkaidoU2012 120919 colop3 pBAD-RBS-eCFP-RBS-AG43-dT-pSTV28(pSB1C3).jpg|thumb|Colony PCR result]]
 +
 +
We noticed the some of colones have pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 construct as plasmid DNA.
 +
 +
<br style="line-height: 0; clear: both;" />
 +
</div></div>
<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
==September 19th==
+
===September 20th===
-
<div>
+
<div class="hokkaidou-section">
-
==Colony PCR of pBAD-RBS-eCFP-RBS-Ag43-dI-pSB1C3==
+
-
<p>
+
 +
====Digestion of ptet-RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 (DNA solution eluted from colony No.10, 12 respectively in ) and ptet-pSB1A2====
 +
To make a construct of ptet-RBS-phaC-RBS-phaA-RBS-phaB-RBS-dT-pSB1A2, we digested RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 with XbaI and PstI, ptet-pSB1A2 with SpeI and PstI. We prepared DNa solution derived from No. 10 and No. 12 colony selected selected by the result of colony PCR for 18th.
 +
<br />
 +
Insert No.10 (RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2)
 +
{|class="hokkaidou-table-digestion"
 +
  |-
 +
  |DNA solution (40 ng/ul)
 +
  |20 ul
 +
  |-
 +
  |EcoRI
 +
  |1 ul
 +
  |-
 +
  |PstI
 +
  |1 ul
 +
  |-
 +
  |10xH buffer
 +
  |4 ul
 +
  |-
 +
  |DW
 +
  |4 ul
 +
  |-
 +
  |Total
 +
  |30 ul
 +
  |}
 +
 +
 +
Insert No.12 (RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2)
 +
{|class="hokkaidou-table-digestion"
 +
  |-
 +
  |DNA solution (40 ng/ul)
 +
  |20 ul
 +
  |-
 +
  |EcoRI
 +
  |1 ul
 +
  |-
 +
  |PstI
 +
  |1 ul
 +
  |-
 +
  |10xH buffer
 +
  |4 ul
 +
  |-
 +
  |DW
 +
  |4 ul
 +
  |-
 +
  |Total
 +
  |30 ul
 +
  |}
 +
 +
 +
Vector(ptet-pSB1A2)
 +
{|class="hokkaidou-table-digestion"
 +
  |-
 +
  |DNA solution (about 40 ng/ul)
 +
  |2 ul
 +
  |-
 +
  |EcoRI
 +
  |1 ul
 +
  |-
 +
  |PstI
 +
  |1 ul
 +
  |-
 +
  |10xH buffer
 +
  |2 ul
 +
  |-
 +
  |DW
 +
  |14 ul
 +
  |-
 +
  |Total
 +
  |20 ul
 +
  |}
 +
 +
 +
{|class="hokkaidou-table-digestion"
 +
|-
 +
|Number
 +
|Degree
 +
|Minute
 +
|-
 +
|1
 +
|37
 +
|120
 +
|-
 +
|2
 +
|60
 +
|15
 +
|-
 +
|3
 +
|4
 +
|HOLD
 +
|}
 +
 +
 +
[[image:HokkaidoU2012 120919 digestion InsertNo10-InsertNo12-Vector.jpg|thumb|digestion result]]
 +
 +
====Ethanol precipitation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2====
 +
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
 +
#Centrifuged at 15000 rpm, 15 min at 4C.
 +
#Removed supernatant and added 220 ul of 70% ethanol.
 +
#Centrifuged at 15000 rpm, 10 min at 4C.
 +
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW.
 +
 +
 +
[[image:HokkaidoU2012 120919 Ethapre InsertNo10-InsertNo12-Vector.jpg|thumb|ethanol precipitation result]]
 +
We decided to use No. 10 digestion product for ligation, and confirmed that the concentration of Insert DNA solution is 50 ng/ul and Vector DNA solution is about 20~30 ng/ul.
 +
 +
====Ligation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2====
 +
{|class="hokkaidou-table-ligation"
 +
|-
 +
|Insert DNA (50 ng/ul)
 +
|4 ul
 +
|-
 +
|Vector DNA (20~30 ng/ul)
 +
|1.5 ul
 +
|-
 +
|DW
 +
|0.5 ul
 +
|-
 +
|Ligation Mighty Mix
 +
|6 ul
 +
|-
 +
|Total
 +
|12 ul
 +
|}
 +
 +
 +
Ligation reaction time was in detail below.
 +
 +
{|class="hokkaidou-table-ligation"
 +
|-
 +
|Degree
 +
|Minute
 +
|-
 +
|16
 +
|30
 +
|-
 +
|65
 +
|10
 +
|-
 +
|4
 +
|Hold
 +
|}
 +
 +
 +
====Transformation of RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2====
 +
Transformation of ligation product into JM109.
 +
#Mixed 2 ul RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 ligation product to 50 ul of thawed competent cells on ice.
 +
#Incubated on ice for 30 min.
 +
#Mixed 350 ul of LB. 
 +
#Incubated for 2 hrs to get the resistance to Chloramphenicol.
 +
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBC).
 +
#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.
 +
#Incubated the plates at 37C for 15 hrs.
 +
 +
<br style="line-height: 0; clear: both;" />
 +
 +
</div></div>
 +
 +
<div class="hokkaidou-notebook-daily">
 +
===September 21th===
 +
<div class="hokkaidou-section">
 +
====Colony PCR of pBAD-RBS-eCFP-RBS-Ag43-dI-pSB1C3====
{|class="hokkaidou-table-pcr-reagent"
{|class="hokkaidou-table-pcr-reagent"
Line 386: Line 655:
|10 ul
|10 ul
|-
|-
-
|Forward Primer(200bp down primer)
+
|Forward Primer(X-phaB-F primer)
|0.8 ul
|0.8 ul
|-
|-
-
|Reverse Primer(ag43-f4 primer)
+
|Reverse Primer(PS-R primer)
|0.8 ul
|0.8 ul
|-
|-
Line 415: Line 684:
|-
|-
|3
|3
-
|53.0
+
|68.9
|30
|30
|-
|-
Line 433: Line 702:
   
   
-
We used N1 (DW only) and N2(pBAD-RBS-Ag43-dT-pSB1A2)as controls.
+
We used N1 (DW only) and N2(RBS-phaC-RBs-phaA-RBS-phaB-pSB1A2)as controls.
-
Desired product is about 702bp.
+
Desired product is about 1500bp.
 +
 
 +
[[image:HokkaidoU2012 120921 colop1 ptet-RBS-phaCAB-RBS-eYFP-dT-pSB1C3.jpg|thumb|Colony PCR result]]
 +
[[image:HokkaidoU2012 120921 colop2 ptet-RBS-phaCAB-RBS-eYFP-dT-pSB1C3.jpg|thumb|Colony PCR result2]]
-
[[image:|thumb|Colony PCR result]]
+
Target products exist in almost all samples. We selected No. 1, 2 colony for incubation for plasmid extraction and No. 6, 12, 14 for storing at 4C. 
-
There are some desired products exist in plated colonies. We selected No.5,6 for Incubation for mini-prep and No.10,11 for stock at 4C.
+
<br style="line-height: 0; clear: both;" />
-
</p>
+
</div></div>
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Latest revision as of 03:24, 27 September 2012

Contents

September 17th

Digestion of RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 and ptet-pSB1A2

To make a construct of ptet-RBS-phaC-RBS-phaA-RBS-phaB-RBS-dT-pSB1A2, we digested RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 with XbaI and PstI, ptet-pSB1A2 with SpeI and PstI.
Insert (RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2)

DNA solution (45~50 ng/ul) 27 ul
XbaI 1 ul
PstI 1 ul
10xM buffer 4 ul
DW 7 ul
Total 40 ul


Vector(ptet-pSB1A2)

DNA solution (about 20 ng/ul) 4 ul
SpeI 1 ul
PstI 1 ul
10xH buffer 2 ul
DW 12 ul
Total 20 ul


Number Degree Minute
1 37 120
2 60 15
3 4 HOLD


Number Degree Minute
1 37 120
2 70 20
3 4 HOLD


Ethanol precipitation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2

  1. Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
  2. Centrifuged at 15000 rpm, 15 min at 4C.
  3. Removed supernatant and added 220 ul of 70% ethanol.
  4. Centrifuged at 15000 rpm, 10 min at 4C.
  5. Removed supernatant and dried out at room temperature, after that added 10 ul of DW.

We confirmed that the concentration of Insert DNA solution is 60~70 ng/ul and Vector DNA solution is about 20~30 ng/ul.

Ligation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2

Insert DNA (60~70 ng/ul) 4 ul
Vector DNA (20~30 ng/ul) 2 ul
Ligation Mighty Mix 6 ul
Total 12 ul


Ligation reaction time was in detail below.

Degree Minute
16 30
65 10
4 Hold

Transformation of RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2

Transformation of ligation ligation product into JM109.

  1. Mixed 2 ul RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-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 17 hrs.


September 18th

Colony PCR of pBAD-RBS-eCFP-RBS-Ag43-dI-pSB1C3

We did colony PCR two times.

DNA solution 4 ul
Kapa-Taq(Taq polymerase) 10 ul
Forward Primer(200bp down primer) 0.8 ul
Reverse Primer(ag43-f4 primer) 0.8 ul
DW 4.4 ul
Total 20 ul


Number Degree Second
1 95 120
2 95 30
3 53.0 30
4 72 60
5 72 60
6 4 HOLD

Cycle:2~4 x 35


We used N1 (DW only) and N2(pBAD-RBS-Ag43-dT-pSB1A2)as controls. Desired product is about 702bp.

Colony PCR result
Colony PCR result 2

Target products didn't exist in all samples. We noticed the reason why such results shown is that we used incorrect pSB1C3 DNA solution which isn't confirmed the sequence. We'll try the synthesis once more.

Digestion of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 and pSB1C3

To make a construct of pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3, we digested pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 with EcoRI and SpeI and pSB1C3 with EcoRI and SpeI.
Insert (pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28)

DNA solution (about 35ng/ul) 41 ul
EcoRI 2 ul
SpeI 2 ul
10xH buffer 5 ul
Total 50 ul


Vector(pSB1C3)

DNA solution (about 30~40 ng/ul) 2 ul
EcoRI 1 ul
SpeI 1 ul
10xH buffer 2 ul
DW 14 ul
Total 20 ul


Number Degree Minute
1 37 120
2 60 15
3 4 HOLD


digestion result

Ethanol precipitation of pBAD-RBS-eCFP-RBS-Ag43-dT--pSB1C3

  1. Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
  2. Centrifuged at 15000 rpm, 15 min at 4C.
  3. Removed supernatant and added 220 ul of 70% ethanol.
  4. Centrifuged at 15000 rpm, 10 min at 4C.
  5. Removed supernatant and dried out at room temperature, after that added 10 ul of DW.
ethanol precipitation result

Ligation of pBAD-RBS-eCFP-RBS-Ag43-dT and pSB1C3

Insert DNA 4 ul
Vector DNA 2 ul
Ligation Mighty Mix 6 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-Ag43-dT-pSB1C3

Transformation ligation product into JM109.

  1. Mixed 2 ul pBAD-RBS-eCFP-RBS-Ag43-dT--pSB1C3 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. Incubated for 2 hrs to get the resistance to Chloramphenicol.
  5. Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBC).
  6. Plated 300 ul of the culture onto first dish and spread.
  7. Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.
  8. Incubated the plates at 37C for 15 hrs.


September 19th

Colony PCR of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28

Colony PCR to confirm whether pSTV28 vettor has pBAS-RBs-eCFP-RBs-Ag43-dT as insert or not.

DNA solution 4 ul
Kapa-Taq(Taq polymerase) 10 ul
Forward Primer(200bp down primer) 0.8 ul
Reverse Primer(ag43-f4 primer) 0.8 ul
DW 4.4 ul
Total 20 ul


Number Degree Second
1 95 120
2 95 30
3 53.3 30
4 72 180
5 72 60
6 4 HOLD

Cycle:2~4 x 35


We used N1 (DW only) and N2(pBAD-RBS-Ag43-dT-pSB1A2)as controls. Desired product is about 702bp.

Colony PCR result

We noticed the some of colones have pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 construct as plasmid DNA.

Colony PCR of pBAD-RBS-eCFP-RBS-Ag43-dI-pSB1C3 and pBAD-RBS-eCFP-RBS-Ag43-dI on pSB1C3

Colony PCR to confirm whether the constructs written above really have eCFP and Ag43 coding sites or not by using primers: one can anneal to a part of eCFP coding site and another can anneal to a part of Ag43 coding site.

DNA solution 4 ul
Kapa-Taq(Taq polymerase) 10 ul
Forward Primer(FP-F primer) 0.8 ul
Reverse Primer(ag43-R primer) 0.8 ul
DW 4.4 ul
Total 20 ul


Number Degree Second
1 95 120
2 95 30
3 53.3 30
4 72 180
5 72 60
6 4 HOLD

Cycle:2~4 x 35

We used N1, N2 (DW only) as controls. Desired product is about 452 bp and 697 bp respectively.

Colony PCR result

We noticed the some of colones have pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 construct as plasmid DNA.


September 20th

Digestion of ptet-RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 (DNA solution eluted from colony No.10, 12 respectively in ) and ptet-pSB1A2

To make a construct of ptet-RBS-phaC-RBS-phaA-RBS-phaB-RBS-dT-pSB1A2, we digested RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 with XbaI and PstI, ptet-pSB1A2 with SpeI and PstI. We prepared DNa solution derived from No. 10 and No. 12 colony selected selected by the result of colony PCR for 18th.
Insert No.10 (RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2)

DNA solution (40 ng/ul) 20 ul
EcoRI 1 ul
PstI 1 ul
10xH buffer 4 ul
DW 4 ul
Total 30 ul


Insert No.12 (RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2)

DNA solution (40 ng/ul) 20 ul
EcoRI 1 ul
PstI 1 ul
10xH buffer 4 ul
DW 4 ul
Total 30 ul


Vector(ptet-pSB1A2)

DNA solution (about 40 ng/ul) 2 ul
EcoRI 1 ul
PstI 1 ul
10xH buffer 2 ul
DW 14 ul
Total 20 ul


Number Degree Minute
1 37 120
2 60 15
3 4 HOLD


digestion result

Ethanol precipitation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2

  1. Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
  2. Centrifuged at 15000 rpm, 15 min at 4C.
  3. Removed supernatant and added 220 ul of 70% ethanol.
  4. Centrifuged at 15000 rpm, 10 min at 4C.
  5. Removed supernatant and dried out at room temperature, after that added 10 ul of DW.


ethanol precipitation result

We decided to use No. 10 digestion product for ligation, and confirmed that the concentration of Insert DNA solution is 50 ng/ul and Vector DNA solution is about 20~30 ng/ul.

Ligation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2

Insert DNA (50 ng/ul) 4 ul
Vector DNA (20~30 ng/ul) 1.5 ul
DW 0.5 ul
Ligation Mighty Mix 6 ul
Total 12 ul


Ligation reaction time was in detail below.

Degree Minute
16 30
65 10
4 Hold


Transformation of RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2

Transformation of ligation product into JM109.

  1. Mixed 2 ul RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-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. Incubated for 2 hrs to get the resistance to Chloramphenicol.
  5. Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBC).
  6. Plated 300 ul of the culture onto first dish and spread.
  7. Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.
  8. Incubated the plates at 37C for 15 hrs.


September 21th

Colony PCR of pBAD-RBS-eCFP-RBS-Ag43-dI-pSB1C3

DNA solution 4 ul
Kapa-Taq(Taq polymerase) 10 ul
Forward Primer(X-phaB-F primer) 0.8 ul
Reverse Primer(PS-R primer) 0.8 ul
DW 4.4 ul
Total 20 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(RBS-phaC-RBs-phaA-RBS-phaB-pSB1A2)as controls. Desired product is about 1500bp.

Colony PCR result
Colony PCR result2

Target products exist in almost all samples. We selected No. 1, 2 colony for incubation for plasmid extraction and No. 6, 12, 14 for storing at 4C.



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