Team:KIT-Kyoto/c6h12o6

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Revision as of 12:02, 25 September 2012

August 1st

*TNFAIP3

Since it takes at least one month to establish transgenic flies, we decided to first construct the P-element plasmid for microinjection into Drosophia embryos.

① We performed PCR in the following conditions.

primer

(ⅰ)TNFAIP3
Composition for reaction

	sample1
10ng/μL TNFAIP3	6μL
10× KOD plus buffer	10μL
2mM dNTPs	10μL
25mM MgSO₄	3.2μL
10P 5'primer	3μL
10P 3'primer	3μL
KOD plus	2μL
dH₂O	62.8μL
Total	100μL

Reaction conditions

temperature	time	cycle
95°C	2min.
95°C	15sec	25cycle
60°C	30sec	25cycle
68°C	2min30sec	25cycle
68°C	2min30sec
14°C	∞

August 2nd

*TNFA and API2

① PCR product check

We run 0.7% agarose gel electorophoresis in the following conditions. Composition

	8/1 the attB TNFAIP3 PCR products
DNA sample	10μL
6×Dye	2μL
total	12μL

The order of sample application
Left : 1kb marker(5uL)
Right: attB TNFAIP3
Results

②　DNA　purification from gel
We electrophoresed in 0.7% agarose gel in the following conditions.

Composition

	8/1 attB TNFAIP3 PCRproduct
DNA sample	90μL
6×Dye	18μL
total	108μL

We applied half of 108 uL of sample to each of two lanes of Agaroe gel with 5uL of 1kb marker.Then we detected the DNA band in the gel.

Results

We purifed DNA from the gel by QIA Quick Gel Extraction Kit.

・Eatimation of the amount of attB TNFAIP3 DNA fragments by electrophoresis.

Order of samples applied to the agarose gel
The samples were electrophoresed in 0.7% agarose gel in following order.

Right to left:3uL of 1kb marker 1uL of 5 fold dilution of attB TNFAIP3 DNA fragments,1uL of 10 fold dilution of attB TNFAIP3 DNA fragments, 1uL of 20 fold dilution of attB TNFAIP3 DNA fragments, 1uL of 1kb marker.

Results

We have estimated amount of the attB TNFAIP3 DNA to be 80ng/uL.

Result

August 3rd

*TNFA and API2

attB TNFAIP3(80ng/μL)	1μL
pDONR(455ng/μL)	0.35μL
TE buffer	7.65
total	9μL

August 4th

*TNFA and API2

August 5th

*TNFA and API2

August 6th

*TNFA and API2

August 7th

*TNFA and API2

	pDONR	BP TNFA-1	BP TNFA-2
DNA sample	1μL	1μL	1μL
6×Dye	1μL	1μL	1μL
dH₂O	4μL	4μL	4μL
total	6μL	6μL	6μL

BP TNFA-2	2μL
pTFW(Destination vector)	0.5μL
TE buffer	6.5μL
total	9μL

WEEK１終わり

August 8th

*TNFA and API2

August 9th

*TNFA and API2

DNA sample	1μL
10×H buffer	0.5μL
EcoRⅠ	0.2μL
dH₂O	3.3μL
total	5μL

	1-2
50ng/μL LR TNFA-1	1μL
10×rTaq buffer	2μL
2mM dNTPs	2μL
25mM MgCl₂	0.8μL
10P 5'primer	0.6μL
10P 3'primer	0.6μL
rTaq	0.4μL
dH₂O	12.6μL
total	20μL

temperature	time	cycle
95°C	2min
95°C	15sec	25cycle
55°C	30sec	25cycle
68°C	2min30sec	25cycle
68°C	2min30sec
14°C	∞

August 10th

*TNFA and API2

August 13th

*TNFA and API2

1-1	1μL
10×M buffer	0.5μL
Hind Ⅲ	0.2μL
dH₂O	3.3μL
total	5μL

August 14th

*TNFA and API2

1-1	sample1
10ng/μL TNFAIP3	6μL
10×KOD plus buffer	10μL
2mM dNTPs	10μL
25mM MgSO₄	3.2μL
10P 5'primer	3μL
10P 3'primer	3μL
KOD plus	2μL
dH₂O	62.8μL
total	100μL

temperature	time	cycle
95°C	2min
95°C	15sec	25cycle
60°C	30sec	25cycle
68°C	2min30sec	25cycle
68°C	2min30sec
14°C	∞

WEEK2終わり

August 20th

TNFA and API2

1. Reproduction of DNA from gel
We did agarose gel electrophoresis (0.7% gel).

Composition

	a product of PCR attB TNFAIP3(8/1)
DNA sample	90μL
6×Dye	18μL
Total	108μL

We separated 54uL×2 from 108uL (sample) and we did electrophoresis (54uL sample and 1kb marker 5uL).We separated gel containing DNA.

Results
We isolated DNA from these gels by QIA quick Gel Extraction Kit.
Finally we melt DNA to TE Buffer(40uL)

August 21st

TNFA and API2

1. Density measurement of attB TNFAIP3
We did agarose gel electrophoresis (0.7% gel). (1kb marker-3uL, 5times dilution attB TNFAIP3-1uL (make August 20 ), 10times dilution-1uL, 1kb marker-2uL, 15times dilution-1uL, 20times dilution-1uL, 1kb marker-1uL )

Results
We estimated attB TNFAIP3(we make this time) is 35ng/uL

2. BP reaction
We adjusted solution (vials) on 1.5mL tube to next composition.

attB TNFAIP3(35ng/μL)	2μL
pDONR(150ng/μL)	1μL
TE buffer	5μL
Total	8μL

We added BP Clonase Ⅱ enzyme mix-2uL to this vials.
We incubated these vials for 2hour at 25˚C. Next we did BP reaction

3. Transformation
We added BP TNFAIP3-2uL to XL1-Blue-100uL and we did Transformation.
Finally we spread 10uL, 40uL and 100uL on plate of LB Kanamycin(＋) and we keep 37℃ overnight.

August 22nd

TNFA and API2

We separated six colonies from 40uL spread plate of BP TNFAIP3 (make August 21 ) .
We cultivated these colonies on 2.5mL Kanamycin(＋) LB culture medium and we kept shaking this medium overnight.

@@ Line 10: / Line 10: @@
 <br>
 <br>
-Composition
+Since it takes at least one month to establish transgenic flies, we decided to first construct the P-element plasmid for microinjection into Drosophia embryos.
+<br>
+<br>
+① We performed PCR in the following conditions.<br>
+<br>
+<strong>primer</strong><br>
+<br>
+(ⅰ)TNFAIP3<br>
+Composition for reaction
 <Table Border Cellspacing="0">
 <Tr><Td>　　</Td><Td>　sample1　</Td></Tr>
@@ Line 21: / Line 30: @@
 <Tr><Td>　KOD plus　</Td><Td>　2μL　</Td></Tr>
 <Tr><Td>　dH<sub>2</sub>O　</Td><Td>　62.8μL　</Td></Tr>
-<Tr><Td>　total　</Td><Td>　100μL　</Td></Tr>
+<Tr><Td>　Total　</Td><Td>　100μL　</Td></Tr>
 </Table>
 <br>
+Reaction conditions
 <Table Border Cellspacing="0">
 <Tr><Td>　temperature　</Td><Td>　time　</Td><Td>　cycle　</Td></Tr>
-<Tr><Td>　95°C　</Td><Td>　2min　</Td><Td>　　</Td></Tr>
+<Tr><Td>　95°C　</Td><Td>　2min.　</Td><Td>　　</Td></Tr>
 <Tr><Td>　95°C　</Td><Td>　15sec　</Td><Td>　25cycle　</Td></Tr>
 <Tr><Td>　60°C　</Td><Td>　30sec　</Td><Td>　25cycle　</Td></Tr>
@@ Line 43: / Line 52: @@
 <strong>*TNFA and API2</strong>
 <br><br>
+① PCR product check<br>
+<br>
+We run 0.7% agarose gel electorophoresis in the following conditions.
 Composition
 <Table Border Cellspacing="0">
-<Tr><Td>　　</Td><Td>　1-2　</Td></Tr>
+<Tr><Td>　　</Td><Td>　8/1 the attB TNFAIP3 PCR products　</Td></Tr>
 <Tr><Td>　DNA sample　</Td><Td>　10μL　</Td></Tr>
 <Tr><Td>　6×Dye　</Td><Td>　2μL　</Td></Tr>
 <Tr><Td>　total　</Td><Td>　12μL　</Td></Tr>
-</Table>
+</Table><br>
 <br>
+The order of sample application<br>
+Left : 1kb marker(5uL)<br>
+Right: attB TNFAIP3<br>
+<strong>Results</strong>
 <br>
 <img src="https://static.igem.org/mediawiki/2012/e/e0/0802.png" width="500" height="300">
 <br>
+<br>
+②　DNA　purification from gel<br>
+ We electrophoresed in 0.7% agarose gel in the following conditions.<br>
 <br>
 Composition
 <Table Border Cellspacing="0">
-<Tr><Td>　　</Td><Td>　1-2　</Td></Tr>
+<Tr><Td>　　</Td><Td>　8/1 attB TNFAIP3 PCRproduct　</Td></Tr>
 <Tr><Td>　DNA sample　</Td><Td>　90μL　</Td></Tr>
 <Tr><Td>　6×Dye　</Td><Td>　18μL　</Td></Tr>
 <Tr><Td>　total　</Td><Td>　108μL　</Td></Tr>
 </Table>
+<br><br>
+We applied half of 108 uL of sample to each of two lanes of Agaroe gel with 5uL of 1kb marker.Then we detected the DNA band in the gel.<br><br>
+<strong>Results</strong><br>
+<img src="https://static.igem.org/mediawiki/2012/1/19/0802-2.png" width="500" height="300">
 <br>
+We purifed DNA from the gel by QIA Quick Gel Extraction Kit.<br>
+<br>
+・Eatimation of the amount of attB TNFAIP3 DNA fragments by electrophoresis.<br>
+<br>
+Order of samples applied to the agarose gel<br>
+The samples were electrophoresed in 0.7% agarose gel in following order.<br>
+<br>
+Right to left:3uL of 1kb marker 1uL of 5 fold dilution of attB TNFAIP3 DNA fragments,1uL of 10 fold dilution of attB TNFAIP3 DNA fragments, 1uL of 20 fold dilution of attB TNFAIP3 DNA fragments, 1uL of 1kb marker.<br>
 <br>
-<img src="https://static.igem.org/mediawiki/2012/1/19/0802-2.png" width="500" height="300">
+<strong>Results</strong>
-<br><br>
+<br>
 <img src="https://static.igem.org/mediawiki/2012/0/0e/0802-3.png" width="500" height="300">
 <br>
+We have estimated amount of the attB TNFAIP3 DNA to be 80ng/uL.<br><br>
 <br>