Team:HokkaidoU Japan/Notebook/plastic Week 9

From 2012.igem.org

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Cycle:2~4 x 35
Cycle:2~4 x 35
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<br />
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{|class="hokkaidou-table-pcr-reagent"
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|Solution
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|Volume(ul)
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|-
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|DNA
 +
|1
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|-
 +
|Suffix-EX (10uM)
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|2
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|-
 +
|Prefix-PS (10uM)
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|2
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|-
 +
|KAPA Taq
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|25
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|-
 +
|DW
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|20
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|Total
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|50
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{|class="hokkaidou-table-pcr-time"
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|-
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|Number
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|Degree
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|Second
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|-
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|1
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|95
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|120
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|2
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|95
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|30
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|-
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|3
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|63.7
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|30
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|-
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|4
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|72
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|180
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|-
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|5
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|4
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|HOLD
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|}
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Cycle:2~4 x 35
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Revision as of 09:32, 29 August 2012

Contents

August 27th

Digestion

Digestion to divide BBa_K342001(PhaC) with XbaI and PstI.
And BBa_B0034(RBS) with 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



Gel extraction

Gel extraction for digestion product. 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.
Because the length of pSB1C3 is nearly PhaC.
And I digested PhaC(BBa_K342001) and pSB1C3 with 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


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 ethanolysis　of PHB polymer for 4hrs 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 63.7 30
4 68 60
5 4 HOLD ↑STEP DOWNに書き換える

Cycle:2~4 x 35

Solution Volume(ul)
DNA 1
Suffix-EX (10uM) 2
Prefix-PS (10uM) 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

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