Team:HokkaidoU Japan/Notebook/aggregation Week 4

From 2012.igem.org

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<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
==July 23th==
+
===July 23rd===
-
<div>
+
<div class="hokkaidou-section">
-
==Mini-prep==
+
====Plasmid extraction====
-
<p>
+
Plasmid extraction for Ag43(resuspended colony incubated at July 17th and resuspended colony incubated at July 20th). We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.  
-
mini-prep for Ag43(7/17 cultivated colony resuspended and 7/20 cultivated colony resuspended). We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50ul of DNA solutions.  
+
-
</p>
+
 +
<br style="line-height: 0; clear: both;" />
</div></div>
</div></div>
 +
<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
==July 24th==
+
===July 24th===
-
<div>
+
<div class="hokkaidou-section">
-
==Electrophoresis==
+
=====Electrophoresis=====
-
<p>
+
Electrophoresis for Ag43(plasmid extraction at July 23rd) and Ag43 digestion results(digested with EcoRI and SpeI)
-
Electrophoresis for Ag43(mini-preped above) and Ag43 digestion results(digested with EcoRI and SpeI)
+
[[image:HokkaidoU2012 120724 ag43-dt pcr.jpg|thumb|Plasmid extraction result]]
-
 
+
-
[[image:HokkaidoU2012 120724 ag43-dt pcr.jpg|thumb|Mini-prep result]]
+
-
 
+
[[image:HokkaidoU2012 120724 Ag43(K346007 cut with E&amp;S) digestion product.jpg|thumb|Digestion result]]
[[image:HokkaidoU2012 120724 Ag43(K346007 cut with E&amp;S) digestion product.jpg|thumb|Digestion result]]
 +
From this digestion result, we found out that one or two enzymes didn't work successfully but there are enough concentration of DNA in 3000bp band to use for digestion.
-
In this digestion result, we knew that one or two enzymes didn't work successfully but there are enough concentration of DNA in 3000bp band to use in digestion.
+
====Gel extraction====
 +
Gel extraction for digestion production. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.  
-
</p>
+
====Ethanol precipitation====
-
==Gel extraction==
+
-
<p>
+
-
Gel extraction for digestion production. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50ul of DNA solution.
+
-
</p>
+
-
==Ethanol precipitation==
+
-
<p>
+
Ethanol precipitation for digestion and gel extraction product.
Ethanol precipitation for digestion and gel extraction product.
-
#Added 5ul of NaoAc, 1.5ul of glycogen and 125ul of 100% ethanol.
+
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
-
#Centrifuged in 15000rpm, 10min at 4C.
+
#Centrifuged at 15000 rpm, 10min at 4C.
-
#Remove supernatant and added 220ul of 70% ethanol.
+
#Removed supernatant and added 220 ul of 70% ethanol.
-
#Centrifuged in 15000rpm, 10min at 4C.
+
#Centrifuged at 15000 rpm, 10 min at 4C.
-
#Remove supernatant and air drying in room temperature then added 10ul of DW.  
+
#Removed supernatant and dried out at room temperature after that added 10 ul of DW.  
[[image:HokkaidoU2012 120724 Ag43(K346007 cut with E&amp;S) digestion product ethanol precipitation.jpg|thumb|Ethanol precipitation result]]
[[image:HokkaidoU2012 120724 Ag43(K346007 cut with E&amp;S) digestion product ethanol precipitation.jpg|thumb|Ethanol precipitation result]]
-
In this result, we estimated that the concentration of ethanol precipitation product is about 40ng/ul.
+
From this result, we estimated that the concentration of ethanol precipitation product is about 40 ng/ul.
-
 
+
-
</p>
+
-
==Digestion==
+
-
<p>
+
-
Digestion to confirm how many PstI cutting sites are in K346007(Ag43 coading) and Ag43-dT complex digestion with SpeI and XbaI.
+
-
 
+
 +
====Digestion====
 +
Digestion to confirm how many PstI cutting sites are there in K346007 and for Ag43-dT complex with SpeI and XbaI.
 +
<br />
Ag43
Ag43
PstI
PstI
Line 54: Line 44:
   |-
   |-
   |DNA solution
   |DNA solution
-
   |5ul
+
   |5 ul
   |-
   |-
   |PstI
   |PstI
-
   |1ul
+
   |1 ul
   |-
   |-
   |10xH buffer
   |10xH buffer
-
   |2ul
+
   |2 ul
   |-
   |-
   |DW
   |DW
-
   |12ul
+
   |12 ul
   |-
   |-
   |Total
   |Total
-
   |20ul
+
   |20 ul
   |}
   |}
-
Ag43-dT
+
Ag43-dT<br />
SpeI and XbaI
SpeI and XbaI
  {|class="hokkaidou-table-digestion"
  {|class="hokkaidou-table-digestion"
   |-
   |-
   |DNA solution
   |DNA solution
-
   |12ul
+
   |12 ul
   |-
   |-
   |SpeI
   |SpeI
-
   |1ul
+
   |1 ul
   |-
   |-
   |XbaI
   |XbaI
-
   |1ul
+
   |1 ul
   |-
   |-
   |10xH buffer
   |10xH buffer
-
   |2ul
+
   |2 ul
   |-
   |-
   |DW
   |DW
-
   |4ul
+
   |4 ul
   |-
   |-
   |Total
   |Total
-
   |20ul
+
   |20 ul
   |}
   |}
-
</p>
+
 
-
==Electrophoresis==
+
====Electrophoresis====
-
<p>
+
[[image:HokkaidoU2012 120724 Ag43(K346007 cut with E&amp;S then cut with P).jpg|thumb|Ag43 d+(P) Digestion result]]
 +
[[image:HokkaidoU2012 120724 ag43-dplus.jpg|thumb|Ag43-dT d+(X&S) Digestion result]]
Electrophoresis for digestion results.
Electrophoresis for digestion results.
-
[[image:|thumb|Ag43 d+(P) Digestion result]]
+
From this result, we found that '''there are 6 PstI cutting sites in K346007(Ag43)'''.
-
[[image:|thumb|Ag43-dT d+(X&S) Digestion result]]
+
-
</p>
+
====Gel extraction====
-
==Gel extraction==
+
Gel extraction of Ag43-dt digestion result. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.
-
<p>
+
 
-
Gel ectraction of Ag43-dt digestio result.We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50ul of DNA solution.
+
====Digestion====
-
</p>
+
Ag43-dT and pSB1AK3 mixture solution(each DNA fragment is about 3kbp) digested with HindIII to digest pSB1AK3.
-
+
 
 +
{|class="hokkaidou-table-digestion"
 +
  |-
 +
  |DNA solution
 +
  |8 ul
 +
  |-
 +
  |HindIII
 +
  |1 ul
 +
  |-
 +
  |10xM buffer
 +
  |1 ul
 +
  |-
 +
  |Total
 +
  |10 ul
 +
  |}
 +
 
 +
<br style="line-height: 0; clear: both;" />
</div></div>
</div></div>
 +
 +
<div class="hokkaidou-notebook-daily">
 +
===July 25th===
 +
<div class="hokkaidou-section">
 +
====Digestion====
 +
Digestion of pT7-RBS on pSB1K3 with SpeI.
 +
 +
{|class="hokkaidou-table-digestion"
 +
  |-
 +
  |DNA solution
 +
  |3 ul
 +
  |-
 +
  |SpeI
 +
  |1 ul
 +
  |-
 +
  |10xH buffer
 +
  |1 ul
 +
  |-
 +
  |DW
 +
  |5 ul
 +
  |-
 +
  |Total
 +
  |10 ul
 +
  |}
 +
[[image:HokkaidoU2012 120725 Digestion Ag43-dt on pSB1AK3(HindIII) &amp; pT7-RBS on pSB1K3(SpeI).jpg|thumb|Digestion result(Ag43-dT and pT7-RBS)]]
 +
We were confirmed that pSB1AK3 was digested and became 1.3k and 1.8k bp fragments by HindIII. 
 +
 +
====Gel extraction====
 +
Gel extraction of Ag43-dT on pSB1AK3(HindIII) and pT7-RBS on pSB1K3(SpeI).
 +
We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.
 +
 +
====Ethanol precipitation====
 +
Ethanol precipitation of digestion and gel extraction products.
 +
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
 +
#Centrifuged at 15000 rpm, 10 min at 4C.
 +
#Removed supernatant and added 220 ul of 70% ethanol.
 +
#Centrifuged at 15000 rpm, 5 min at 4C.
 +
#Removed supernatant and dried out at room temperature after that added 5 ul of DW.
 +
 +
====Ligation====
 +
Ligation for pT7-RBS on pSB1K3(Vector) and Ag43-dT(Insert).
 +
{|class="hokkaidou-table-ligation"
 +
|-
 +
|pT7-RBS on pSB1K3
 +
|2 ul
 +
|-
 +
|Ag43-dT
 +
|2 ul
 +
|-
 +
|DW
 +
|1 ul
 +
|-
 +
|Ligation Mighty Mix(TAKARA)
 +
|5 ul
 +
|-
 +
|Total
 +
|10 ul
 +
|}
 +
 +
 +
{|class="hokkaidou-table-ligation"
 +
|-
 +
|Degree
 +
|Minute
 +
|-
 +
|16
 +
|30
 +
|-
 +
|65
 +
|10
 +
|-
 +
|4
 +
|Hold
 +
|}
 +
 +
<br style="line-height: 0; clear: both;" />
 +
</div></div>
 +
 +
<div class="hokkaidou-notebook-daily">
 +
===July 26th===
 +
<div class="hokkaidou-section">
 +
====Transformation====
 +
Transformation plasmid DNA ligated at 25th (pT7-RBS-Ag43-dT on pSB1K3) into BL21.
 +
#Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.
 +
#Incubated on ice for 30 min.
 +
#Added 200 ul of LB then incubated the cells for 2 hrs at 37C.
 +
#Prepared and Labeled two petri dishes with LBK.
 +
#Spread 200 ul of the transformation onto first dish.
 +
#Added 450 ul of LB to 50 ul of the transformation and spread 200 ul of it onto second dish.
 +
#Incubated the plates at 37C for over 30 hrs.
 +
 +
There were no colony on the plates.
 +
 +
====Electrophoresis====
 +
Electrophoresis of digestion and ligation products.
 +
#Placed TBE agarose gel in Electrophoresis chamber.
 +
#Added 1/2X TBE buffer to Electrophoresis chamber.
 +
#Added 5 ul of EtBr and ran at 100 V for 30 min.
 +
#Apply 1kb DNA ladder and each samples.
 +
#Ran at 100 V for 30 min.
 +
[[image:HokkaidoU2012 120726 digestion ligation.jpg|thumb|Digestion and Ligation results]]
 +
There are no band in the lane of ligation products. But if digestion products were not ligated, two bands of digestion products would exist in the lane. 
 +
 +
<br style="line-height: 0; clear: both;" />
 +
</div></div>
 +
 +
<div class="hokkaidou-notebook-daily">
 +
===July 27th===
 +
<div class="hokkaidou-section">
 +
====Transformation====
 +
Transformation plasmid DNA ligated at 25th (pT7-RBS-Ag43-dT on pSB1K3) into BL21.
 +
#Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.
 +
#Incubated on ice for 30 min.
 +
#Added 600 ul of LB then incubated the cells for 2 hrs at 37C.
 +
#Prepared and Labeled three petri dishes with LBK X2 and LBC.
 +
#Spread 300 ul of the transformation onto LBK dish.
 +
#Added 900 ul of LB to 100 ul of the transformation and spread 300 ul of it onto LBC dish and LBK dish.
 +
#Incubated the plates at 37C for 17 hrs and 30 min.
 +
 +
====Ligation====
 +
Ligation of pT7-RBS on pSB1K3(Vector) and Ag43-dT(Insert)
 +
{|class="hokkaidou-table-ligation"
 +
|-
 +
|pT7-RBS on pSB1K3
 +
|2 ul
 +
|-
 +
|Ag43-dT
 +
|2 ul
 +
|-
 +
|DW
 +
|1 ul
 +
|-
 +
|Ligation Mighty Mix(TAKARA)
 +
|5 ul
 +
|-
 +
|Total
 +
|10 ul
 +
|}
 +
 +
 +
{|class="hokkaidou-table-ligation"
 +
|-
 +
|Degree
 +
|Minute
 +
|-
 +
|16
 +
|30
 +
|-
 +
|65
 +
|10
 +
|-
 +
|4
 +
|Hold
 +
|}
 +
 +
<br style="line-height: 0; clear: both;" />
 +
</div></div>
 +
 +
<div class="hokkaidou-notebook-daily">
 +
===July 28th===
 +
<div class="hokkaidou-section">
 +
[[image:HokkaidoU2012 120728 Transformation result.jpg|thumb|Transformation result left:LBC center:LBK right:LBK]]
 +
There were many colonies on LBC. We guessed we mistook pT7-RBS on pSB1C3 for pT7-RBS on pSB1K3.
 +
 +
====Liquid culture====
 +
Liquid culture for pT7-RBS-Ag43-dT on pSB1C3.
 +
#Added 2 ml of LBC into culture tubes.
 +
#Resuspended 5 colonies.
 +
#Incubated the tubes at 30C for 22 hrs.
 +
 +
====Digestion====
 +
Digestion of pT7-RBS on pSB1K3 with SpeI and Ag43-dT on pSB1AK3(cut with SpeI & XbaI) with HindIII.
 +
<br />
 +
pT7-RBS on pSB1K3
 +
{|class="hokkaidou-table-digestion"
 +
  |-
 +
  |DNA solution
 +
  |3 ul
 +
  |-
 +
  |SpeI
 +
  |1 ul
 +
  |-
 +
  |10xH buffer
 +
  |1 ul
 +
  |-
 +
  |DW
 +
  |5 ul
 +
  |-
 +
  |Total
 +
  |10 ul
 +
  |}
 +
 +
Ag43-dT on pSB1AK3(cut with SpeI & XbaI)
 +
{|class="hokkaidou-table-digestion"
 +
  |-
 +
  |DNA solution
 +
  |8 ul
 +
  |-
 +
  |HindIII
 +
  |1 ul
 +
  |-
 +
  |10xM buffer
 +
  |1 ul
 +
  |-
 +
  |Total
 +
  |10 ul
 +
  |}
 +
 +
Digested at 37C for 2 hrs.
 +
[[image:HokkaidoU2012 120728 Digestion pT7-RBS on pSB1K3(SpeI) Ag43-dT on pSB1AK3(HindIII).jpg|thumb|digestion result]]
 +
This results showed that pSB1AK3 was successfully digested into fragments(1.3K and 1.8K bp), but we couldn't confirm whether pT7-RBS on pSB1K3 was digested or not.
 +
 +
====Gel extraction====
 +
Gel extraction for digestion production. We used FastGene Gel&PCR extraction kit(NipponGenetics) and got 50 ul of DNA solution.
 +
 +
====Ethanol precipitation====
 +
Ethanol precipitation for digestion and gel extraction product.
 +
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
 +
#Centrifuged at 15000 rpm, 10 min at 4C.
 +
#Removed supernatant and added 220 ul of 70% ethanol.
 +
#Centrifuged at 15000 rpm, 10 min at 4C.
 +
#Removed supernatant and dryed out at room temperature after that added 10 ul of DW.
 +
 +
====Ligation====
 +
Ligation of pT7-RBS on pSB1K3(Vector) and Ag43-dT(Insert)
 +
 +
{|class="hokkaidou-table-ligation"
 +
|-
 +
|pT7-RBS on pSB1K3
 +
|2 ul
 +
|-
 +
|Ag43-dT
 +
|2 ul
 +
|-
 +
|DW
 +
|1 ul
 +
|-
 +
|Ligation Mighty Mix(TAKARA)
 +
|5 ul
 +
|-
 +
|Total
 +
|10 ul
 +
|}
 +
 +
 +
{|class="hokkaidou-table-ligation"
 +
|-
 +
|Degree
 +
|Minute
 +
|-
 +
|16
 +
|30
 +
|-
 +
|65
 +
|10
 +
|-
 +
|4
 +
|Hold
 +
|}
 +
 +
<br style="line-height: 0; clear: both;" />
 +
</div></div>
 +
 +
<div class="hokkaidou-notebook-daily">
 +
===July 29th===
 +
<div class="hokkaidou-section">
 +
====Plasmid extraction====
 +
Plasmid extraction for 5 cultures of pT7-RBS-Ag43-dT. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.
 +
[[image:HokkaidoU2012_120729_pt7-rbs-ag43-dt.jpg|thumb|Plasmid extraction products]]
 +
 +
====Digestion====
 +
We need to confirm the DNA is pT7-RBS-Ag43-dT or not.<br />
 +
Plasmid extraction products(pT7-RBS-Ag43-dT)
 +
PstI
 +
{|class="hokkaidou-table-digestion"
 +
  |-
 +
  |DNA solution
 +
  |4 ul
 +
  |-
 +
  |PstI
 +
  |1 ul
 +
  |-
 +
  |10xH buffer
 +
  |2 ul
 +
  |-
 +
  |DW
 +
  |13 ul
 +
  |-
 +
  |Total
 +
  |20 ul
 +
  |}
 +
 +
 +
Plasmid extraction products(pT7-RBS-Ag43-dT)<br />
 +
EcoR1 and Spe1
 +
{|class="hokkaidou-table-digestion"
 +
  |-
 +
  |DNA solution
 +
  |4 ul
 +
  |-
 +
  |EcoR1
 +
  |1 ul
 +
  |-
 +
  |Spe1
 +
  |1 ul
 +
  |-
 +
  |10xH buffer
 +
  |2 ul
 +
  |-
 +
  |DW
 +
  |12 ul
 +
  |-
 +
  |Total
 +
  |20 ul
 +
  |}
 +
 +
====Electrophoresis====
 +
Electrophoresis for digestion results.
 +
[[image:HokkaidoU2012_120729_ag43-dig.jpg|thumb|Digestion result]]
 +
I could not understand what is happend.
 +
I tried it again, but the result was the same.
 +
 +
<br style="line-height: 0; clear: both;" />
 +
</div></div>
 +
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi -->
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi -->
</div>
</div>
<br style="line-height: 0; clear: both;" />
<br style="line-height: 0; clear: both;" />
{{Team:HokkaidoU_Japan/footer}}
{{Team:HokkaidoU_Japan/footer}}

Latest revision as of 03:18, 27 September 2012

Contents

July 23rd

Plasmid extraction

Plasmid extraction for Ag43(resuspended colony incubated at July 17th and resuspended colony incubated at July 20th). We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.


July 24th

Electrophoresis

Electrophoresis for Ag43(plasmid extraction at July 23rd) and Ag43 digestion results(digested with EcoRI and SpeI)

Plasmid extraction result
Digestion result

From this digestion result, we found out that one or two enzymes didn't work successfully but there are enough concentration of DNA in 3000bp band to use for digestion.

Gel extraction

Gel extraction for digestion production. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.

Ethanol precipitation

Ethanol precipitation for digestion and gel extraction product.

  1. Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
  2. Centrifuged at 15000 rpm, 10min 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

From this result, we estimated that the concentration of ethanol precipitation product is about 40 ng/ul.

Digestion

Digestion to confirm how many PstI cutting sites are there in K346007 and for Ag43-dT complex with SpeI and XbaI.
Ag43 PstI

DNA solution 5 ul
PstI 1 ul
10xH buffer 2 ul
DW 12 ul
Total 20 ul


Ag43-dT
SpeI and XbaI

DNA solution 12 ul
SpeI 1 ul
XbaI 1 ul
10xH buffer 2 ul
DW 4 ul
Total 20 ul


Electrophoresis

Ag43 d+(P) Digestion result
Ag43-dT d+(X&S) Digestion result

Electrophoresis for digestion results.

From this result, we found that there are 6 PstI cutting sites in K346007(Ag43).

Gel extraction

Gel extraction of Ag43-dt digestion result. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.

Digestion

Ag43-dT and pSB1AK3 mixture solution(each DNA fragment is about 3kbp) digested with HindIII to digest pSB1AK3.

DNA solution 8 ul
HindIII 1 ul
10xM buffer 1 ul
Total 10 ul


July 25th

Digestion

Digestion of pT7-RBS on pSB1K3 with SpeI.

DNA solution 3 ul
SpeI 1 ul
10xH buffer 1 ul
DW 5 ul
Total 10 ul
Digestion result(Ag43-dT and pT7-RBS)

We were confirmed that pSB1AK3 was digested and became 1.3k and 1.8k bp fragments by HindIII.

Gel extraction

Gel extraction of Ag43-dT on pSB1AK3(HindIII) and pT7-RBS on pSB1K3(SpeI). We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.

Ethanol precipitation

Ethanol precipitation of digestion and gel extraction products.

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

Ligation

Ligation for pT7-RBS on pSB1K3(Vector) and Ag43-dT(Insert).

pT7-RBS on pSB1K3 2 ul
Ag43-dT 2 ul
DW 1 ul
Ligation Mighty Mix(TAKARA) 5 ul
Total 10 ul


Degree Minute
16 30
65 10
4 Hold


July 26th

Transformation

Transformation plasmid DNA ligated at 25th (pT7-RBS-Ag43-dT on pSB1K3) into BL21.

  1. Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.
  2. Incubated on ice for 30 min.
  3. Added 200 ul of LB then incubated the cells for 2 hrs at 37C.
  4. Prepared and Labeled two petri dishes with LBK.
  5. Spread 200 ul of the transformation onto first dish.
  6. Added 450 ul of LB to 50 ul of the transformation and spread 200 ul of it onto second dish.
  7. Incubated the plates at 37C for over 30 hrs.

There were no colony on the plates.

Electrophoresis

Electrophoresis of digestion and ligation products.

  1. Placed TBE agarose gel in Electrophoresis chamber.
  2. Added 1/2X TBE buffer to Electrophoresis chamber.
  3. Added 5 ul of EtBr and ran at 100 V for 30 min.
  4. Apply 1kb DNA ladder and each samples.
  5. Ran at 100 V for 30 min.
Digestion and Ligation results

There are no band in the lane of ligation products. But if digestion products were not ligated, two bands of digestion products would exist in the lane.


July 27th

Transformation

Transformation plasmid DNA ligated at 25th (pT7-RBS-Ag43-dT on pSB1K3) into BL21.

  1. Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.
  2. Incubated on ice for 30 min.
  3. Added 600 ul of LB then incubated the cells for 2 hrs at 37C.
  4. Prepared and Labeled three petri dishes with LBK X2 and LBC.
  5. Spread 300 ul of the transformation onto LBK dish.
  6. Added 900 ul of LB to 100 ul of the transformation and spread 300 ul of it onto LBC dish and LBK dish.
  7. Incubated the plates at 37C for 17 hrs and 30 min.

Ligation

Ligation of pT7-RBS on pSB1K3(Vector) and Ag43-dT(Insert)

pT7-RBS on pSB1K3 2 ul
Ag43-dT 2 ul
DW 1 ul
Ligation Mighty Mix(TAKARA) 5 ul
Total 10 ul


Degree Minute
16 30
65 10
4 Hold


July 28th

Transformation result left:LBC center:LBK right:LBK

There were many colonies on LBC. We guessed we mistook pT7-RBS on pSB1C3 for pT7-RBS on pSB1K3.

Liquid culture

Liquid culture for pT7-RBS-Ag43-dT on pSB1C3.

  1. Added 2 ml of LBC into culture tubes.
  2. Resuspended 5 colonies.
  3. Incubated the tubes at 30C for 22 hrs.

Digestion

Digestion of pT7-RBS on pSB1K3 with SpeI and Ag43-dT on pSB1AK3(cut with SpeI & XbaI) with HindIII.
pT7-RBS on pSB1K3

DNA solution 3 ul
SpeI 1 ul
10xH buffer 1 ul
DW 5 ul
Total 10 ul

Ag43-dT on pSB1AK3(cut with SpeI & XbaI)

DNA solution 8 ul
HindIII 1 ul
10xM buffer 1 ul
Total 10 ul

Digested at 37C for 2 hrs.

digestion result

This results showed that pSB1AK3 was successfully digested into fragments(1.3K and 1.8K bp), but we couldn't confirm whether pT7-RBS on pSB1K3 was digested or not.

Gel extraction

Gel extraction for digestion production. We used FastGene Gel&PCR extraction kit(NipponGenetics) and got 50 ul of DNA solution.

Ethanol precipitation

Ethanol precipitation for digestion and gel extraction product.

  1. Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
  2. Centrifuged at 15000 rpm, 10 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 dryed out at room temperature after that added 10 ul of DW.

Ligation

Ligation of pT7-RBS on pSB1K3(Vector) and Ag43-dT(Insert)

pT7-RBS on pSB1K3 2 ul
Ag43-dT 2 ul
DW 1 ul
Ligation Mighty Mix(TAKARA) 5 ul
Total 10 ul


Degree Minute
16 30
65 10
4 Hold


July 29th

Plasmid extraction

Plasmid extraction for 5 cultures of pT7-RBS-Ag43-dT. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.

Plasmid extraction products

Digestion

We need to confirm the DNA is pT7-RBS-Ag43-dT or not.
Plasmid extraction products(pT7-RBS-Ag43-dT) PstI

DNA solution 4 ul
PstI 1 ul
10xH buffer 2 ul
DW 13 ul
Total 20 ul


Plasmid extraction products(pT7-RBS-Ag43-dT)
EcoR1 and Spe1

DNA solution 4 ul
EcoR1 1 ul
Spe1 1 ul
10xH buffer 2 ul
DW 12 ul
Total 20 ul

Electrophoresis

Electrophoresis for digestion results.

Digestion result

I could not understand what is happend. I tried it again, but the result was the same.



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