Team:HokkaidoU Japan/Notebook/aggregation Week 6

From 2012.igem.org

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<!-- DO NOT EDIT OVER THIS LINE @iTakeshi -->
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi -->
<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
==August 6th==
+
===August 6th===
-
<div>
+
<div class="hokkaidou-section">
-
==Ethanol precipitation==
+
====Ethanol precipitation====
-
<p>
+
Ethanol precipitation for digestion and gel extraction product.
Ethanol precipitation for digestion and gel extraction product.
#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.
Line 14: Line 13:
#Centrifuged at 15000 rpm, 10 min at 4C.
#Centrifuged at 15000 rpm, 10 min at 4C.
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW.  
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW.  
-
</p>
 
-
==Ligation==
+
====Ligation====
-
<p>
+
We ligated pT7-RBS on pSB1K3 (digested Spe1) as vector and Ag43-dT (digested Spe1 and Xba1 and HindIII) as insert.
We ligated pT7-RBS on pSB1K3 (digested Spe1) as vector and Ag43-dT (digested Spe1 and Xba1 and HindIII) as insert.
-
 
{|class="hokkaidou-table-ligation"
{|class="hokkaidou-table-ligation"
Line 38: Line 34:
|10 ul
|10 ul
|}
|}
-
 
Ligation reaction time was written below.
Ligation reaction time was written below.
-
 
{|class="hokkaidou-table-ligation"
{|class="hokkaidou-table-ligation"
|-
|-
Line 56: Line 50:
|Hold
|Hold
|}
|}
-
</p>
 
-
==Plasmid extraction==
+
 
-
<p>
+
====Plasmid extraction====
Plasmid extraction of pBad-RBS. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.
Plasmid extraction of pBad-RBS. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.
[[image:HokkaidoU2012_120806_pBad-rbs.jpg|thumb|Plasmid extraction result]]
[[image:HokkaidoU2012_120806_pBad-rbs.jpg|thumb|Plasmid extraction result]]
-
</p>
 
-
==Transformation==
+
====Transformation====
-
<p>
+
Transformation of plasmid DNA (pT7-RBS-Ag43-dT on pSB1K3) into DH5&alpha;.
-
Transformation of plasmid DNA (pT7-RBS-Ag43-dT on pSB1K3) in DH5&alpha;.
+
#Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.
#Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.
#Incubated on ice for 30 min.
#Incubated on ice for 30 min.
Line 75: Line 66:
#Incubated them at 37C for 16 hours.
#Incubated them at 37C for 16 hours.
-
</p>
+
====Ethanol precipitation====
-
 
+
-
==Ethanol precipitation==
+
-
<p>
+
Ethanol precipitation for plasmid extraction product (pBad-RBS). Because the refine of plasmid extraction product is not enough. <br />
Ethanol precipitation for plasmid extraction product (pBad-RBS). Because the refine of plasmid extraction product is not enough. <br />
We used 15 ul of all solution.
We used 15 ul of all solution.
Line 86: Line 74:
#Centrifuged at 15000 rpm, 5 min at 4C.
#Centrifuged at 15000 rpm, 5 min at 4C.
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW.   
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW.   
-
</p>
 
-
==Digestion==
+
====Digestion====
-
<p>
+
Digestion of ethanol precipitation product(pBad-RBS).
Digestion of ethanol precipitation product(pBad-RBS).
  {|class="hokkaidou-table-digestion"
  {|class="hokkaidou-table-digestion"
Line 108: Line 94:
   |10 ul
   |10 ul
   |}
   |}
-
</p>
 
-
[[image:HokkaidoU2012_120807_pbad-rbs-digested.jpg|thumb|digestion result]]
 
 +
[[image:HokkaidoU2012_120807_pbad-rbs-digested.jpg|thumb|digestion result]]
From this result, speI digested DNA completely.<br />
From this result, speI digested DNA completely.<br />
So I think that the pT7-RBS on pSB1K3 DNA solution is not pure.
So I think that the pT7-RBS on pSB1K3 DNA solution is not pure.
</div></div>
</div></div>
-
 
<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
==August 7th==
+
===August 7th===
-
<div>
+
<div class="hokkaidou-section">
-
==Gel extraction==
+
====Gel extraction====
-
<p>
+
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.  
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.  
-
</p>
 
-
==Ethanol precipitation==
+
====Ethanol precipitation====
-
<p>
+
Ethanol precipitation for digestion and gel extraction product.
Ethanol precipitation for digestion and gel extraction product.
#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.
Line 132: Line 113:
#Centrifuged at 15000 rpm, 10 min at 4C.
#Centrifuged at 15000 rpm, 10 min at 4C.
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW.  
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW.  
-
</p>
 
-
==Colony PCR==
+
====Colony PCR====
-
<p>
+
Colony PCR to confirm that whether the Ag43-dT was successfully ligated with pT7-RBS on pSB1K3 or not.   
Colony PCR to confirm that whether the Ag43-dT was successfully ligated with pT7-RBS on pSB1K3 or not.   
-
 
-
 
{|class="hokkaidou-table-pcr-reagent"
{|class="hokkaidou-table-pcr-reagent"
|-
|-
Line 192: Line 169:
We used N1 (DW only) and N2 (Ag43 on pSB1C3 (K346007)) as controls.
We used N1 (DW only) and N2 (Ag43 on pSB1C3 (K346007)) as controls.
Desired product is about 500~600bp.
Desired product is about 500~600bp.
-
 
[[image:HokkaidoU2012_120807_colonyPCR.jpg|thumb|Colony PCR result]]
[[image:HokkaidoU2012_120807_colonyPCR.jpg|thumb|Colony PCR result]]
-
 
We thought that colonies of No. 1 and 2 and 5 and 11 and 14 have deep band.
We thought that colonies of No. 1 and 2 and 5 and 11 and 14 have deep band.
Next step, we resuspended these 5 colonies and cultured (add 1700 ul LB and 2 ul Amp) for 15 hours at 37C.
Next step, we resuspended these 5 colonies and cultured (add 1700 ul LB and 2 ul Amp) for 15 hours at 37C.
-
 
-
</p>
 
</div></div>
</div></div>
-
 
-
 
<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
==August 8th==
+
===August 8th===
-
<div>
+
<div class="hokkaidou-section">
-
==Plasmid extraction==
+
====Plasmid extraction====
-
<p>
+
Plasmid extraction of colony No. 1 and 14. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.
Plasmid extraction of colony No. 1 and 14. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.
[[image:HokkaidoU2012_120808_pt7-rbs-ag43-dt.jpg|thumb|Plasmid extraction result]]
[[image:HokkaidoU2012_120808_pt7-rbs-ag43-dt.jpg|thumb|Plasmid extraction result]]
And then, we did plasmid extraction for colony No. 2,5,11. The results of them are the same.
And then, we did plasmid extraction for colony No. 2,5,11. The results of them are the same.
-
</p>
 
-
==Sequencing==
+
====Sequencing====
-
<p>
+
PCR for sequencing.
PCR for sequencing.
-
 
{|
{|
|DNA
|DNA
Line 310: Line 277:
To purify the PCR product, we did ethanol precipitation.
To purify the PCR product, we did ethanol precipitation.
-
 
Ethanol precipitation for sequencing.
Ethanol precipitation for sequencing.
#Added 10 ul of H2O, 2 ul of NaoAc, 1 ul of glycogen and 50 ul of 100% ethanol.
#Added 10 ul of H2O, 2 ul of NaoAc, 1 ul of glycogen and 50 ul of 100% ethanol.
Line 317: Line 283:
#Centrifuged at 15000 rpm, 5 min at 26C.
#Centrifuged at 15000 rpm, 5 min at 26C.
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW.  
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW.  
-
 
Then ran a sequencing machine (ByoDye Terminator v3.1 Cycle Sequencing Kit)
Then ran a sequencing machine (ByoDye Terminator v3.1 Cycle Sequencing Kit)
-
</p>
 
</div></div>
</div></div>
-
 
<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
==August 9th==
+
===August 9th===
-
<div>
+
<div class="hokkaidou-section">
-
==Ligation==
+
====Ligation====
-
<p>
+
We ligated pT7-RBS on pSB1K3 (digested Spe1) as vector and Ag43-dT (digested Spe1 and Xba1 and HindIII) as insert.<br />
We ligated pT7-RBS on pSB1K3 (digested Spe1) as vector and Ag43-dT (digested Spe1 and Xba1 and HindIII) as insert.<br />
We did ligation (pT7-RBS on pSB1K3 and Ag43-dT) and transformation it, at August 6th.
We did ligation (pT7-RBS on pSB1K3 and Ag43-dT) and transformation it, at August 6th.
Line 351: Line 313:
Ligation reaction time was written below.
Ligation reaction time was written below.
-
 
{|class="hokkaidou-table-ligation"
{|class="hokkaidou-table-ligation"
|-
|-
Line 366: Line 327:
|Hold
|Hold
|}
|}
-
</p>
 
-
==Transformation==
+
====Transformation====
-
<p>
+
Transformation of plasmid DNA ligation product (pT7-RBS-Ag43-dT on pSB1K3) into DH5&alpha;.<br />
-
Transformation of plasmid DNA ligation product (pT7-RBS-Ag43-dT on pSB1K3) in DH5&alpha;.<br />
+
Transformation of August 6th, we found only 14 colonies. So this time, we use 3 ul of DNA solution for transformation.
Transformation of August 6th, we found only 14 colonies. So this time, we use 3 ul of DNA solution for transformation.
-
 
#Added 3 ul of plasmid DNA to 50 ul of thawed competent cells on ice.
#Added 3 ul of plasmid DNA to 50 ul of thawed competent cells on ice.
#Incubated on ice for 30 min.
#Incubated on ice for 30 min.
Line 380: Line 338:
#Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.  
#Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.  
#Incubated them at 37C for 16 hours.
#Incubated them at 37C for 16 hours.
-
</p>
 
-
 
</div></div>
</div></div>
-
 
<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
==August 11st==
+
===August 11st===
-
<div>
+
<div class="hokkaidou-section">
-
==Colony PCR==
+
====Colony PCR====
-
<p>
+
Colony PCR to confirm that whether the Ag43-dT was successfully ligated with pT7-RBS on pSB1K3 or not.   
Colony PCR to confirm that whether the Ag43-dT was successfully ligated with pT7-RBS on pSB1K3 or not.   
-
 
-
 
{|class="hokkaidou-table-pcr-reagent"
{|class="hokkaidou-table-pcr-reagent"
|-
|-
Line 446: Line 398:
We used N1 (DW only) and N2 (Ag43-dT on pSB1AK3) as controls.
We used N1 (DW only) and N2 (Ag43-dT on pSB1AK3) as controls.
Desired product is about 500~600bp.
Desired product is about 500~600bp.
-
 
[[image:HokkaidoU1120811 coloP.jpg|thumb|Colony PCR result]]
[[image:HokkaidoU1120811 coloP.jpg|thumb|Colony PCR result]]
We thought that colonies of No. 6 and 8 is like N2 band.
We thought that colonies of No. 6 and 8 is like N2 band.
Next step, we resuspended these 2 colonies and cultured (add 1700 ul LB and 2 ul Amp) for 16 hours at 37C.
Next step, we resuspended these 2 colonies and cultured (add 1700 ul LB and 2 ul Amp) for 16 hours at 37C.
-
 
-
</p>
 
-
 
</div></div>
</div></div>
-
 
<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
==August 12nd==
+
===August 12nd===
-
<div>
+
<div class="hokkaidou-section">
-
==Plasmid extraction==
+
====Plasmid extraction====
-
<p>
+
Plasmid extraction of No. 6 and 8. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.
Plasmid extraction of No. 6 and 8. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.
[[image:HokkaidoU2012_120812_pt7-dt.jpg|thumb|plasmid extraction result]]
[[image:HokkaidoU2012_120812_pt7-dt.jpg|thumb|plasmid extraction result]]
-
</p>
+
</div></div>
-
</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 02:26, 27 September 2012

Contents

August 6th

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 dried out at room temperature, after that added 10 ul of DW.

Ligation

We ligated pT7-RBS on pSB1K3 (digested Spe1) as vector and Ag43-dT (digested Spe1 and Xba1 and HindIII) as insert.

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

Ligation reaction time was written below.

Degree Minute
16 30
65 10
4 Hold


Plasmid extraction

Plasmid extraction of pBad-RBS. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.

Plasmid extraction result

Transformation

Transformation of plasmid DNA (pT7-RBS-Ag43-dT on pSB1K3) into DH5α.

  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 hours at 37C.
  4. Prepared and Labeled two LBK plates.
  5. Plated 300 ul of the culture onto first dish and spread.
  6. Added 900 ul of LB to 100 ul of the culture and plated 300 ul of it onto second dish and spread.
  7. Incubated them at 37C for 16 hours.

Ethanol precipitation

Ethanol precipitation for plasmid extraction product (pBad-RBS). Because the refine of plasmid extraction product is not enough.
We used 15 ul of all solution.

  1. Added 1.5 ul of NaoAc(3 M), 1.5 ul of glycogen and 38 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 10 ul of DW.

Digestion

Digestion of ethanol precipitation product(pBad-RBS).

DNA solution 3 ul
SpeI 1 ul
10xM buffer 1 ul
DW 5 ul
Total 10 ul
digestion result

From this result, speI digested DNA completely.
So I think that the pT7-RBS on pSB1K3 DNA solution is not pure.

August 7th

Gel extraction

Gel extraction for digestion product. 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 dried out at room temperature, after that added 10 ul of DW.

Colony PCR

Colony PCR to confirm that whether the Ag43-dT was successfully ligated with pT7-RBS on pSB1K3 or not.

DNA solution 4 ul (1colony/10 ul DW)
Kapa-Taq(Taq polymerase) 5 ul
Forward Primer(Ag43-f4 primer) 0.5 ul
Reverse Primer(PS-R primer) 0.5 ul
Total 10 ul


Number Degree Second
1 95 120
2 95 30
3 53 30
4 72 60
5 72 60
6 4 HOLD

Cycle:2~4 x 35

We used N1 (DW only) and N2 (Ag43 on pSB1C3 (K346007)) as controls. Desired product is about 500~600bp.

Colony PCR result

We thought that colonies of No. 1 and 2 and 5 and 11 and 14 have deep band. Next step, we resuspended these 5 colonies and cultured (add 1700 ul LB and 2 ul Amp) for 15 hours at 37C.

August 8th

Plasmid extraction

Plasmid extraction of colony No. 1 and 14. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.

Plasmid extraction result

And then, we did plasmid extraction for colony No. 2,5,11. The results of them are the same.

Sequencing

PCR for sequencing.

DNA primer
Ag43 plasmid extraction product 100bp-up forward primer, ag43-f1, ag43-f2, ag43-f3, ag43-f4
K542009 100bp-up forward primer, ag43-f1, ag43-f2, ag43-f3, ag43-f4
pT7-RBS on pSB1K3 100bp-up forward primer
pT7-RBS on pSB1C3 100bp-up forward primer
Ag43-dT on pSB1AK3 100bp-up forward primer, ag43-f1, ag43-f2, ag43-f3, ag43-f4
Ag43-dT on pSB1T3 100bp-up forward primer, ag43-f1, ag43-f2, ag43-f3, ag43-f4
pT7-RBS on pSB1K3 100bp-up forward primer


Sequencing PCR

template DNA 1 ul
Ready Reaction Premix 1 ul
5x Sequencing Buffer 1.5 ul
H2O 5 ul
Primer(1 pmol/ul) 1.5 ul
Total 10 ul


Number Degree Second
1 96 10
2 50 5
3 60 240
4 4 HOLD

Cycle:1~3 x 25


To purify the PCR product, we did ethanol precipitation. Ethanol precipitation for sequencing.

  1. Added 10 ul of H2O, 2 ul of NaoAc, 1 ul of glycogen and 50 ul of 100% ethanol.
  2. Centrifuged at 15000 rpm, 15 min at 26C.
  3. Removed supernatant and added 100ul of 70% ethanol.
  4. Centrifuged at 15000 rpm, 5 min at 26C.
  5. Removed supernatant and dried out at room temperature, after that added 10 ul of DW.

Then ran a sequencing machine (ByoDye Terminator v3.1 Cycle Sequencing Kit)

August 9th

Ligation

We ligated pT7-RBS on pSB1K3 (digested Spe1) as vector and Ag43-dT (digested Spe1 and Xba1 and HindIII) as insert.
We did ligation (pT7-RBS on pSB1K3 and Ag43-dT) and transformation it, at August 6th. However, we could not get the target plasmid. (pT7-RBS-Ag43-dT on pSB1K3) So, we did ligation by using more Insert DNA part.

pT7-RBS 1 ul
Ag43-dT 4 ul
Ligation Mighty Mix(TAKARA) 5 ul
Total 10 ul


Ligation reaction time was written below.

Degree Minute
16 30
65 10
4 Hold

Transformation

Transformation of plasmid DNA ligation product (pT7-RBS-Ag43-dT on pSB1K3) into DH5α.
Transformation of August 6th, we found only 14 colonies. So this time, we use 3 ul of DNA solution for transformation.

  1. Added 3 ul of plasmid DNA to 50 ul of thawed competent cells on ice.
  2. Incubated on ice for 30 min.
  3. Added 400 ul of LB then incubated the cells for 2 hours at 37C.
  4. Prepared and Labeled two LBK plates.
  5. Plated 300 ul of the culture onto first dish and spread.
  6. Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.
  7. Incubated them at 37C for 16 hours.

August 11st

Colony PCR

Colony PCR to confirm that whether the Ag43-dT was successfully ligated with pT7-RBS on pSB1K3 or not.

DNA solution 4 ul (1colony/10 ul DW)
Kapa-Taq(Taq polymerase) 5 ul
Forward Primer(Ag43-f4 primer) 0.5 ul
Reverse Primer(PS-R primer) 0.5 ul
Total 10 ul


Number Degree Second
1 95 120
2 95 30
3 53 30
4 72 60
5 72 60
6 4 HOLD

Cycle:2~4 x 35

We used N1 (DW only) and N2 (Ag43-dT on pSB1AK3) as controls. Desired product is about 500~600bp.

Colony PCR result

We thought that colonies of No. 6 and 8 is like N2 band. Next step, we resuspended these 2 colonies and cultured (add 1700 ul LB and 2 ul Amp) for 16 hours at 37C.

August 12nd

Plasmid extraction

Plasmid extraction of No. 6 and 8. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.

plasmid extraction result


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