Team:HokkaidoU Japan/Notebook/plastic Week 9

From 2012.igem.org

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<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
-
==July 2nd==
+
===August 27th===
-
<div>
+
<div class="hokkaidou-section">
-
 
+
[[image:HokkaidoU2012 120827 phaC digestion sugama.jpg|thumb|digestion result]]
-
==Digestion==
+
[[image:HokaidoU2012 120827 RBS digestion sugama.jpg|thumb|digestion result]]
-
<p>
+
====Digestion====
-
Digestion to divide BBa_K342001(PhaC) with XbaI and PstI.<br />And BBa_B0034(RBS) with SpeI and PstI (with three samples).
+
BBa_K342001(PhaC) was digested by XbaI and PstI.<br />And BBa_B0034(RBS) was digested by SpeI and PstI (with three samples).
-
<br /><br />
+
PhaC (BBa_K342001)
PhaC (BBa_K342001)
  {|class="hokkaidou-table-digestion"
  {|class="hokkaidou-table-digestion"
Line 32: Line 31:
   |20 ul
   |20 ul
   |}
   |}
-
<br />
+
 
 +
 
RBS (BBa_B0034)
RBS (BBa_B0034)
-
<br />
+
N0. 1
-
N0.1
+
  {|class="hokkaidou-table-digestion"
  {|class="hokkaidou-table-digestion"
   |-
   |-
Line 56: Line 55:
   |25 ul
   |25 ul
   |}
   |}
-
<br /><br />
+
 
-
N0.2
+
 
 +
N0. 2
  {|class="hokkaidou-table-digestion"
  {|class="hokkaidou-table-digestion"
   |-
   |-
Line 78: Line 78:
   |25 ul
   |25 ul
   |}
   |}
-
<br />
 
-
N0.3<br />
+
 
 +
N0. 3
  {|class="hokkaidou-table-digestion"
  {|class="hokkaidou-table-digestion"
   |-
   |-
Line 101: Line 101:
   |25 ul
   |25 ul
   |}
   |}
-
</p>
 
-
==Gel extraction==
+
====Electrophoresis====
-
<p>
+
We confirmed success of digestion by electrophoresis.<br/>The DNA was extracted from TBE gel and we got DNA solution.
 +
 
 +
====Gel extraction====
 +
The digestion product was extracted. Used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.
 +
 
 +
</div></div>
 +
 
 +
<div class="hokkaidou-notebook-daily">
 +
===August 28th===
 +
<div class="hokkaidou-section">
 +
====Digestion====
 +
Digestion to divide PhaA and PhaB with XbaI and PstI.<br />I digested PhaC(BBa_K342001) with these restriction sites and also XhoI to divide pSB1C3 into pieces, on which PhaC is, that is because the length of pSB1C3 is nearly PhaC.<br />And we digested PhaC (BBa_K342001) and pSB1C3 by XbaI and SpeI.<br /><br />
 +
PhaA
 +
{|class="hokkaidou-table-digestion"
 +
  |-
 +
  |DNA solution (125 ng/ul)
 +
  |6.6 ul
 +
  |-
 +
  |XbaI
 +
  |1 ul
 +
  |-
 +
  |PstI
 +
  |1 ul
 +
  |-
 +
  |10xM buffer
 +
  |2 ul
 +
  |-
 +
  |DW
 +
  |9.4 ul
 +
  |-
 +
  |Total
 +
  |20 ul
 +
  |}
 +
 
 +
 
 +
PhaB
 +
{|class="hokkaidou-table-digestion"
 +
  |-
 +
  |DNA solution (125 ng/ul)
 +
  |4 ul
 +
  |-
 +
  |XbaI
 +
  |1 ul
 +
  |-
 +
  |PstI
 +
  |1 ul
 +
  |-
 +
  |10xM buffer
 +
  |2 ul
 +
  |-
 +
  |DW
 +
  |12 ul
 +
  |-
 +
  |Total
 +
  |20 ul
 +
  |}
 +
 
 +
 
 +
PhaC(BBa_K342001)
 +
{|class="hokkaidou-table-digestion"
 +
  |-
 +
  |DNA solution (125 ng/ul)
 +
  |10 ul
 +
  |-
 +
  |XbaI
 +
  |1 ul
 +
  |-
 +
  |PstI
 +
  |1 ul
 +
  |-
 +
  |XhoI
 +
  |5.1 ul
 +
  |-
 +
  |10xM buffer
 +
  |2 ul
 +
  |-
 +
  |DW
 +
  |0.9 ul
 +
  |-
 +
  |Total
 +
  |20 ul
 +
  |}
 +
 
 +
====Electrophoresis====
 +
We confirmed success of digestion by electrophoresis.<br/>The DNA was extracted from TBE gel and we got DNA solution.
 +
 
 +
====Gel extraction====
Gel extraction for digestion product. Used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.  
Gel extraction for digestion product. Used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.  
-
</p>
+
</div></div>
 +
 
 +
<div class="hokkaidou-notebook-daily">
 +
===August 29th===
 +
<div class="hokkaidou-section">
 +
====PHB polymer ethanolysis====
 +
We did ethanolysisãof PHB polymer for 4 hrs with sample 2~8.
 +
 
 +
====Preparation for GC/MS====
 +
We did the preparation for GC/MS with sample 2~8.
 +
 
 +
====PCR====
 +
We multiplied pSB1C3 by PCR.
 +
Used two different DNA polymerase, KOD-Plus-Neo and KAPA Taq.
 +
{|class="hokkaidou-table-pcr-reagent"
 +
|Solution
 +
|Volume (ul)
 +
|-
 +
|DNA
 +
|1
 +
|-
 +
|Suffix-EX
 +
|1
 +
|-
 +
|Prefix-PS
 +
|1
 +
|-
 +
|MgSO4
 +
|3
 +
|-
 +
|dNTP
 +
|5
 +
|-
 +
|10x KOD-Plus-Neo Buffer
 +
|5
 +
|-
 +
|KOD-Plus-Neo
 +
|1
 +
|-
 +
|DW
 +
|33
 +
|-
 +
|Total
 +
|50
 +
|}
 +
 
 +
 
 +
{|class="hokkaidou-table-pcr-time"
 +
|-
 +
|Number
 +
|Degree
 +
|Second
 +
|-
 +
|1
 +
|94
 +
|120
 +
|-
 +
|2
 +
|98
 +
|10
 +
|-
 +
|3
 +
|74
 +
|2
 +
|-
 +
|4
 +
|98
 +
|10
 +
|-
 +
|5
 +
|72
 +
|120
 +
|-
 +
|6
 +
|98
 +
|10
 +
|-
 +
|7
 +
|70
 +
|120
 +
|-
 +
|8
 +
|98
 +
|10
 +
|-
 +
|9
 +
|68
 +
|120
 +
|-
 +
|10
 +
|68
 +
|420
 +
|-
 +
|11
 +
|4
 +
|HOLD
 +
|}
 +
Cycle1 : 2~3 x 5
 +
Cycle2 : 4~5 x 5
 +
Cycle3 : 6~7 x 5
 +
Cycle4 : 8~9 x 30
 +
 
 +
 
 +
{|class="hokkaidou-table-pcr-reagent"
 +
|Solution
 +
|Volume (ul)
 +
|-
 +
|DNA
 +
|1
 +
|-
 +
|Suffix-EX (10 uM)
 +
|2
 +
|-
 +
|Prefix-PS (10 uM)
 +
|2
 +
|-
 +
|KAPA Taq
 +
|25
 +
|-
 +
|DW
 +
|20
 +
|-
 +
|Total
 +
|50
 +
|}
 +
 
 +
{|class="hokkaidou-table-pcr-time"
 +
|-
 +
|Number
 +
|Degree
 +
|Second
 +
|-
 +
|1
 +
|95
 +
|120
 +
|-
 +
|2
 +
|95
 +
|30
 +
|-
 +
|3
 +
|63.7
 +
|30
 +
|-
 +
|4
 +
|72
 +
|180
 +
|-
 +
|5
 +
|4
 +
|HOLD
 +
|}
 +
Cycle:2~4 x 35
 +
 
 +
</div></div>
 +
 
 +
<div class="hokkaidou-notebook-daily">
 +
===August 30th===
 +
<div class="hokkaidou-section">
 +
====Ethanol precipitation====
 +
Diegested phaA by XbaI and SpeI and RBS by SpeI were condensed by Ethanol precipitation.
 +
 
 +
====Ligation====
 +
PhaA and RBS, PhaB and RBS, PhaB and pSB1C3 were ligated each other.<br />And the DNA were transformed into bacteria.
 +
 
 +
====Colony PCR====
 +
The length of PhaB on pSB1C3 was confirmed by colony PCR.<br />The result showed PhaB and pSB1C3 didn't ligate correctly.
 +
 
 +
====Liquid Culture====
 +
Incubation of bacteria holds RBS (BBa_B0034) - PhaC (K342001) was started.
 +
 
 +
</div></div>
 +
 
 +
<div class="hokkaidou-notebook-daily">
 +
===August 31st===
 +
<div class="hokkaidou-section">
 +
====Colony PCR====
 +
We confirmed the length of the three constructs that transformed at August 30th.<br />The result showed RBS and PhaA were ligated correctly.<br />So the incubation was started.
 +
[[image:HokkaidoU 120831 RBS phaA coloP edit (2).jpg|thumb|center|450px]]
 +
 
 +
====Plasmid extraction====
 +
Plasmid of RBS-PhaC were extracted.<br />And then we got 50ul DNA solution.
 +
 
 +
====Liquid culture====
 +
Incubation of bacteria holds dT (BBa_B0015) was started.
 +
 
 +
</div></div>
 +
 
 +
<div class="hokkaidou-notebook-daily">
 +
===September 1st===
 +
<div class="hokkaidou-section">
 +
 
 +
====Plasmid extraction====
 +
Plasmids of RBS-PhaA and dT (BBa_B0015) were extracted.<br />And then we got 50ul DNA solution.
 +
[[image:HokkaidoU 120901 dT RBS-PhaA RBS-PhaC mini-prepç£ç© edit.jpg|thumb|center|600px|center|Fig. Extracted plasmids of dT and RBS-PhaA on pSB1A2]]<br />
 +
1: dT on pSB1AK3 (About 3.3kbp)<br />
 +
2 to 4: RBS-PhaA on pSB1A2 (About 3.2kbp)<br />
 +
5 to 7: RBS-PhaC on pSB1A2 (About 4.1kbp)<br />We thought sample 4 is not ideal plasmid and trashed it.
 +
 
 +
====Digestion====
 +
RBS-PhaA was digested by XbaI and SpeI restriction site to ligate with RBS-PhaC digested by SpeI site.
 +
[[image:HokkaidoU 120901 RBS-PhaA RBS-PhaC digestion.jpg]]
 +
*1 and 2 is digested RBS-PhaA on pSB1A2.
 +
*Upper fragment is vector, pSB1A2.
 +
*Lower one is an objective fragment, RBS-PhaA (About 1.2kbp).
 +
*And PhaB and pSB1C3 were digested with XbaI and SpeI site.<br/>We decided to try ligation PhaB with pSB1C3 again.
 +
 
 +
====Electrophoresis====
 +
We confirmed success of digestion by electrophoresis.<br/>The DNA was extracted from TBE gel and we got DNA solution.
 +
</div></div>
 +
 
 +
<div class="hokkaidou-notebook-daily">
 +
 
 +
===September 2nd===
 +
<div class="hokkaidou-section">
 +
 
 +
====Ethanol precipitation====
 +
The digested DNAs, RBS-PhaA (No. 1), RBS-PhaA (No. 2), RBS-PhaC on pSB1A2, PhaB and pSB1C3 were concentrated by Ethanol precipitation.
 +
 
 +
====Ligation====
 +
RBS-PhaA (No. 1 and No. 2) was ligated with RBS-PhaC on pSB1A2.<br/>
 +
And PhaB was taken in pSB1C3.
 +
 
 +
====Transformation====
 +
These ligated DNAs transformed into E. coli (strain: DH5&alpha;).<br/>
 +
And then we spread fungus liquid added LB on LB plates include antibiotics.
</div></div>
</div></div>

Latest revision as of 04:02, 27 September 2012

Contents

August 27th

digestion result
digestion result

Digestion

BBa_K342001(PhaC) was digested by XbaI and PstI.
And BBa_B0034(RBS) was digested by SpeI and PstI (with three samples). PhaC (BBa_K342001)

DNA solution (100 ng/ul) 12.5 ul
XbaI 1 ul
PstI 1 ul
10xM buffer 2 ul
DW 3.5 ul
Total 20 ul


RBS (BBa_B0034) N0. 1

DNA solution (20.3 ng/ul) 14.3 ul
SpeI 1 ul
PstI 1 ul
10xH buffer 2.5 ul
DW 0.2 ul
Total 25 ul


N0. 2

DNA solution (15.6 ng/ul) 18.6 ul
SpeI 1 ul
PstI 1 ul
10xH buffer 2.5 ul
DW 1.9 ul
Total 25 ul


N0. 3

DNA solution (16.9 ng/ul) 17.2 ul
SpeI 1 ul
PstI 1 ul
10xH buffer 2.5 ul
DW 3.3 ul
Total 25 ul

Electrophoresis

We confirmed success of digestion by electrophoresis.
The DNA was extracted from TBE gel and we got DNA solution.

Gel extraction

The digestion product was extracted. Used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.

August 28th

Digestion

Digestion to divide PhaA and PhaB with XbaI and PstI.
I digested PhaC(BBa_K342001) with these restriction sites and also XhoI to divide pSB1C3 into pieces, on which PhaC is, that is because the length of pSB1C3 is nearly PhaC.
And we digested PhaC (BBa_K342001) and pSB1C3 by XbaI and SpeI.

PhaA

DNA solution (125 ng/ul) 6.6 ul
XbaI 1 ul
PstI 1 ul
10xM buffer 2 ul
DW 9.4 ul
Total 20 ul


PhaB

DNA solution (125 ng/ul) 4 ul
XbaI 1 ul
PstI 1 ul
10xM buffer 2 ul
DW 12 ul
Total 20 ul


PhaC(BBa_K342001)

DNA solution (125 ng/ul) 10 ul
XbaI 1 ul
PstI 1 ul
XhoI 5.1 ul
10xM buffer 2 ul
DW 0.9 ul
Total 20 ul

Electrophoresis

We confirmed success of digestion by electrophoresis.
The DNA was extracted from TBE gel and we got DNA solution.

Gel extraction

Gel extraction for digestion product. Used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.

August 29th

PHB polymer ethanolysis

We did ethanolysisãof PHB polymer for 4 hrs with sample 2~8.

Preparation for GC/MS

We did the preparation for GC/MS with sample 2~8.

PCR

We multiplied pSB1C3 by PCR. Used two different DNA polymerase, KOD-Plus-Neo and KAPA Taq.

Solution Volume (ul)
DNA 1
Suffix-EX 1
Prefix-PS 1
MgSO4 3
dNTP 5
10x KOD-Plus-Neo Buffer 5
KOD-Plus-Neo 1
DW 33
Total 50


Number Degree Second
1 94 120
2 98 10
3 74 2
4 98 10
5 72 120
6 98 10
7 70 120
8 98 10
9 68 120
10 68 420
11 4 HOLD

Cycle1 : 2~3 x 5 Cycle2 : 4~5 x 5 Cycle3 : 6~7 x 5 Cycle4 : 8~9 x 30


Solution Volume (ul)
DNA 1
Suffix-EX (10 uM) 2
Prefix-PS (10 uM) 2
KAPA Taq 25
DW 20
Total 50
Number Degree Second
1 95 120
2 95 30
3 63.7 30
4 72 180
5 4 HOLD

Cycle:2~4 x 35

August 30th

Ethanol precipitation

Diegested phaA by XbaI and SpeI and RBS by SpeI were condensed by Ethanol precipitation.

Ligation

PhaA and RBS, PhaB and RBS, PhaB and pSB1C3 were ligated each other.
And the DNA were transformed into bacteria.

Colony PCR

The length of PhaB on pSB1C3 was confirmed by colony PCR.
The result showed PhaB and pSB1C3 didn't ligate correctly.

Liquid Culture

Incubation of bacteria holds RBS (BBa_B0034) - PhaC (K342001) was started.

August 31st

Colony PCR

We confirmed the length of the three constructs that transformed at August 30th.
The result showed RBS and PhaA were ligated correctly.
So the incubation was started.

HokkaidoU 120831 RBS phaA coloP edit (2).jpg

Plasmid extraction

Plasmid of RBS-PhaC were extracted.
And then we got 50ul DNA solution.

Liquid culture

Incubation of bacteria holds dT (BBa_B0015) was started.

September 1st

Plasmid extraction

Plasmids of RBS-PhaA and dT (BBa_B0015) were extracted.
And then we got 50ul DNA solution.

File:HokkaidoU 120901 dT RBS-PhaA RBS-PhaC mini-prepç£ç© edit.jpg
Fig. Extracted plasmids of dT and RBS-PhaA on pSB1A2

1: dT on pSB1AK3 (About 3.3kbp)
2 to 4: RBS-PhaA on pSB1A2 (About 3.2kbp)
5 to 7: RBS-PhaC on pSB1A2 (About 4.1kbp)
We thought sample 4 is not ideal plasmid and trashed it.

Digestion

RBS-PhaA was digested by XbaI and SpeI restriction site to ligate with RBS-PhaC digested by SpeI site. HokkaidoU 120901 RBS-PhaA RBS-PhaC digestion.jpg

  • 1 and 2 is digested RBS-PhaA on pSB1A2.
  • Upper fragment is vector, pSB1A2.
  • Lower one is an objective fragment, RBS-PhaA (About 1.2kbp).
  • And PhaB and pSB1C3 were digested with XbaI and SpeI site.
    We decided to try ligation PhaB with pSB1C3 again.

Electrophoresis

We confirmed success of digestion by electrophoresis.
The DNA was extracted from TBE gel and we got DNA solution.

September 2nd

Ethanol precipitation

The digested DNAs, RBS-PhaA (No. 1), RBS-PhaA (No. 2), RBS-PhaC on pSB1A2, PhaB and pSB1C3 were concentrated by Ethanol precipitation.

Ligation

RBS-PhaA (No. 1 and No. 2) was ligated with RBS-PhaC on pSB1A2.
And PhaB was taken in pSB1C3.

Transformation

These ligated DNAs transformed into E. coli (strain: DH5α).
And then we spread fungus liquid added LB on LB plates include antibiotics.


Retrieved from "http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_Week_9"