Team:HokkaidoU Japan/Notebook/plastic Week 9

From 2012.igem.org

(Difference between revisions)

Revision as of 16:04, 26 September 2012

August 27th

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

digestion result

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 I digested PhaC(BBa_K342001) and pSB1C3 by using 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 ethanolysis　of 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(X/S) and RBS(S) 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 31th

Colony PCR

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

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.

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 sample4 is not ideal plasmid and trashed it.

Digestion

RBS-PhaA was digested with XbaI and SpeI restriction site to ligate with RBS-PhaC digested with SpeI site.

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.

@@ Line 10: / Line 10: @@
 ==Digestion==
 <p>
-BBa_K342001(PhaC) was digested 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)
@@ Line 36: / Line 36: @@
 RBS (BBa_B0034)
 <br />
-N0.1
+N0. 1
   {|class="hokkaidou-table-digestion"
    |-
@@ Line 58: / Line 58: @@
    |}
 <br /><br />
-N0.2
+N0. 2
   {|class="hokkaidou-table-digestion"
    |-
@@ Line 81: / Line 81: @@
 <br />
-N0.3<br />
+N0. 3<br />
   {|class="hokkaidou-table-digestion"
    |-
@@ Line 128: / Line 128: @@
 ==Digestion==
 <p>
-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 I digested PhaC(BBa_K342001) and pSB1C3 with XbaI and SpeI.<br /><br />
+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 I digested PhaC(BBa_K342001) and pSB1C3 by using XbaI and SpeI.<br /><br />
 PhaA
   {|class="hokkaidou-table-digestion"
@@ Line 221: / Line 221: @@
 ==PHB polymer ethanolysis==
 <p>
-We did ethanolysis　of PHB polymer for 4hrs with sample 2~8
+We did ethanolysis　of PHB polymer for 4 hrs with sample 2~8
 </p>
@@ Line 239: / Line 239: @@
 {|class="hokkaidou-table-pcr-reagent"
 |Solution
-|Volume(ul)
+|Volume (ul)
 |-
 |DNA
@@ Line 284: / Line 284: @@
 |-
 |3
-|63.7
+|74
-|30
+|2
 |-
 |4
-|68
+|98
-|60
+|10
 |-
 |5
+|72
+|120
+|-
+|6
+|98
+|10
+|-
+|7
+|70
+|120
+|-
+|8
+|98
+|10
+|-
+|9
+|68
+|120
+|-
+|10
+|68
+|420
+|-
+|11
 |4
-|HOLD ↑STEP DOWNに書き換える
+|HOLD
 |}
-Cycle:2~4 x 35
+Cycle1 : 2~3 x 5
+Cycle2 : 4~5 x 5
+Cycle3 : 6~7 x 5
+Cycle4 : 8~9 x 30
 <br />
 <br />
 {|class="hokkaidou-table-pcr-reagent"
 |Solution
-|Volume(ul)
+|Volume (ul)
 |-
 |DNA
 |1
 |-
-|Suffix-EX (10uM)
+|Suffix-EX (10 uM)
 |2
 |-
-|Prefix-PS (10uM)
+|Prefix-PS (10 uM)
 |2
 |-
@@ Line 444: / Line 472: @@
 ==Ethanol precipitation==
 <p>
-The digested DNAs, RBS-PhaA (No.1), RBS-PhaA (No.2), RBS-PhaC on pSB1A2, PhaB and pSB1C3 were concentrated by 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.
 </p>
 ==Ligation==
 <p>
-RBS-PhaA (No.1 and No.2) was ligated with RBS-PhaC on pSB1A2.<br/>
+RBS-PhaA (No. 1 and No. 2) was ligated with RBS-PhaC on pSB1A2.<br/>
 And PhaB was taken in pSB1C3.
 </p>

Team:HokkaidoU Japan/Notebook/plastic Week 9

From 2012.igem.org

Revision as of 16:04, 26 September 2012

Contents

August 27th

Digestion

Electrophoresis

Gel extraction

August 28th

Digestion

Electrophoresis

Gel extraction

August 29th

PHB polymer ethanolysis

Preparation for GC/MS

PCR

August 30th

Ethanol precipitation

Ligation

Colony PCR

Liquid Culture

August 31th

Colony PCR

Plasmid extraction

Liquid culture

September 1st

Plasmid extraction

Digestion

Electrophoresis

September 2nd

Ethanol precipitation

Ligation

Transformation