Team:KIT-Kyoto/Notebook-week1

From 2012.igem.org

(Difference between revisions)
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<br><br><br><br><br><br><br><br>
+
<h2>August 1st</h2>
-
<br>内容
+
<br>
 +
<br>
 +
 
 +
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>
 +
 
 +
(ⅰ)TNFAIP3<br>
 +
Composition for reaction
 +
<Table Border Cellspacing="0">
 +
<Tr><Td>  </Td><Td> sample1 </Td></Tr>
 +
<Tr><Td> 10ng/μL TNFAIP3 </Td><Td> 6μL </Td></Tr>
 +
<Tr><Td> 10× KOD plus buffer </Td><Td> 10μL </Td></Tr>
 +
<Tr><Td> 2mM dNTPs </Td><Td> 10μL </Td></Tr>
 +
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 3.2μL </Td></Tr>
 +
<Tr><Td> 10P 5'primer </Td><Td> 3μL </Td></Tr>
 +
<Tr><Td> 10P 3'primer </Td><Td> 3μL </Td></Tr>
 +
<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>
 +
</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> 15sec </Td><Td> 25cycle </Td></Tr>
 +
<Tr><Td> 60°C </Td><Td> 30sec </Td><Td> 25cycle </Td></Tr>
 +
<Tr><Td> 68°C </Td><Td> 2min30sec </Td><Td> 25cycle </Td></Tr>
 +
<Tr><Td> 68°C </Td><Td> 2min30sec </Td><Td>  </Td></Tr>
 +
<Tr><Td> 14°C </Td><Td> ∞ </Td><Td>  </Td></Tr>
 +
</Table>
 +
<br>
 +
<br>
 +
 
 +
 
 +
<h2>August 2nd</h2>
 +
<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> 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><br>
 +
<br>
 +
 
 +
The order of sample application<br>
 +
Left : 1kb marker(5uL)<br>
 +
Right: attB TNFAIP3<br>
 +
<br>
 +
 
 +
<strong>Results</strong>
 +
 
 +
<br>
 +
<img src="http://2012.igem.org/wiki/images/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> 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="http://2012.igem.org/wiki/images/1/19/0802-2.png" width="500" height="300">
 +
<br>
 +
 
 +
We purifed DNA from the gel by QIA Quick Gel Extraction Kit.<br>
 +
<br><br>
 +
・Eatimation of the amount of attB TNFAIP3 DNA fragments by electrophoresis.<br>
 +
Order of samples applied to the agarose gel<br>
 +
The samples were electrophoresed in 0.7% agarose gel in following order.<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>
 +
 
 +
<strong>Results</strong>
 +
<br>
 +
<img src="http://2012.igem.org/wiki/images/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>
 +
 
 +
 
 +
<h2>August 3rd</h2>
 +
<br>
 +
<br>
 +
1. BP reaction<br>
 +
<br>
 +
 
 +
BP reaction was carried out in the following conditions.
 +
<Table Border Cellspacing="0">
 +
<Tr><Td> attB TNFAIP3(80ng/μL) </Td><Td> 1μL </Td></Tr>
 +
<Tr><Td> pDONR(455ng/μL) </Td><Td> 0.35μL </Td></Tr>
 +
<Tr><Td> TE buffer </Td><Td> 7.65 </Td></Tr>
 +
<Tr><Td> total </Td><Td> 9μL </Td></Tr>
 +
</Table>
 +
<br>
 +
<br>
 +
One uL of BP Clonase Ⅱ enzyme mix was added to this solution and incubated for 2.5 hours.<br>
 +
<br>
 +
<br>
 +
2. Transformation of E.coli by BP reaction products<br>
 +
2uL of BP TNFA reaction product was added to 100uL of E.coli XL1-Blue to do transform ation.<br>
 +
At last, cells were divided into 10,40,100,and 200 uL,and spreaded on LB Kanamycin(+)plate,Plats wera incubated for 16 hours at 37°C.<br>
 +
<br>
 +
<br>
 +
 
 +
 
 +
<h2>August 4th</h2>
 +
<br><br>
 +
 
 +
Four independent 4 colonies were picked up from the plate incubated from 8/3 as described in ②,then cultured in 2.5mL of LB Kanamycin(+) liquid culture medium for 16 hours.<br>
 +
<br>
 +
<br>
 +
 
 +
 
 +
<h2>August 5th</h2>
 +
<br>
 +
<br>
 +
 
 +
Plasmid DNAs were isolated from E.coli cultured in 4 of LB liquid culture medium which we made on 8/4 by the alkaline-lysis method, we added the DNA to 20uL TE and added 10uL of the solution to 2uL of 6×Dye. Then we electrophoresed it with marker, 2uL of pDONR+ 2uL of 6×Dye+ 8uL of TE,in 0.7% agarose gel in following order (from left to right)<br>
 +
<br>
 +
Left control pDONR DNA, BP TNFA-1,-2,-3,-4<br>
 +
<br>
 +
 
 +
<strong>Results</strong><br>
 +
<img src="http://2012.igem.org/wiki/images/3/31/0805.png" width="500" height="300"><br>
 +
<br>
 +
<br>
 +
 
 +
<h2>August 6th</h2>
 +
<br>
 +
<br>
 +
E.coli carrying the plasmid sample -1 and -2 in LB Kanamycin(+)liquid culture medium for 16 hours.<br>
 +
<br>
 +
<br>
 +
 
 +
<h2>August 7th</h2>
 +
<br>
 +
<br>
 +
1. Purification of candidate pENTR-TNFAIP3<br>
 +
We purified candidate plasmid-1 and -2 by QIA Prep Spin Miniprep Kit, dissolved  it in 30uL of Buffer EB.<br>
 +
Then,the samples were electrophoresed in 0.7% agarose gel.<br>
 +
<br>
 +
 
 +
We made samples as follows.
 +
<Table Border Cellspacing="0">
 +
<Tr><Td>  </Td><Td> pDONR </Td><Td> BP TNFA-1 </Td><Td> BP TNFA-2 </Td></Tr>
 +
<Tr><Td> DNA sample </Td><Td> 1μL </Td><Td> 1μL </Td><Td> 1μL </Td></Tr>
 +
<Tr><Td> 6×Dye </Td><Td> 1μL </Td><Td> 1μL </Td><Td> 1μL </Td></Tr>
 +
<Tr><Td> dH<sub>2</sub>O </Td><Td> 4μL </Td><Td> 4μL </Td><Td> 4μL </Td></Tr>
 +
<Tr><Td> total </Td><Td> 6μL </Td><Td> 6μL </Td><Td> 6μL </Td></Tr>
 +
</Table>
 +
<br>
 +
<br>
 +
We electrophoresed them in following order.<br>
 +
Left marker 5uL control pDONR DNA ,pENTR-TNFAIP3-1 DNA,pENTR-TNFAIP3 -2 DNA marker 4uL marker 3uL Wright.<br>
 +
<br>
 +
<strong>Results</strong><br>
 +
<img src="http://2012.igem.org/wiki/images/2/2e/0807.png" width="500" height="300">
 +
<br>
 +
Concentration of pENTR-TNFAIP3-2 DNA was estimated at around 100ng/uL.<br>
 +
<br>
 +
<br>
 +
2. LR reaction<br>
 +
We made following solution(vials)in 1.5mL tube.<br>
 +
 
 +
<Table Border Cellspacing="0">
 +
<Tr><Td> BP TNFA-2 </Td><Td> 2μL </Td></Tr>
 +
<Tr><Td> pTFW(Destination vector) </Td><Td> 0.5μL </Td></Tr>
 +
<Tr><Td> TE buffer </Td><Td> 6.5μL </Td></Tr>
 +
<Tr><Td> total </Td><Td> 9μL </Td></Tr>
 +
</Table>
 +
<br>
 +
we added 1uL of LR clonaseⅡ enzyme mix to this solution and incubate it for 2.5 hours.<br>
 +
<br>
 +
3. Transformation of E.coli with LR reaction products.<br>
 +
Transformation of E.coli was carried out by adding 2uL of LR reaction products to 100uL of XL1-Blue and plated on the LB ampicillin(+)plate and cultured at 37°C.<br>
 +
<br>
 +
<br>
 +
 
<BR>
<BR>
<div align="right"><a href="http://2012.igem.org/Team:KIT-Kyoto/Notebook-week2">>>>>>>>>>WEEK2</a></div>
<div align="right"><a href="http://2012.igem.org/Team:KIT-Kyoto/Notebook-week2">>>>>>>>>>WEEK2</a></div>

Revision as of 13:14, 25 September 2012






August 1st



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.

(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



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.


August 3rd



1. BP reaction

BP reaction was carried out in the following conditions.
 attB TNFAIP3(80ng/μL)  1μL 
 pDONR(455ng/μL)  0.35μL 
 TE buffer  7.65 
 total  9μL 


One uL of BP Clonase Ⅱ enzyme mix was added to this solution and incubated for 2.5 hours.


2. Transformation of E.coli by BP reaction products
2uL of BP TNFA reaction product was added to 100uL of E.coli XL1-Blue to do transform ation.
At last, cells were divided into 10,40,100,and 200 uL,and spreaded on LB Kanamycin(+)plate,Plats wera incubated for 16 hours at 37°C.


August 4th



Four independent 4 colonies were picked up from the plate incubated from 8/3 as described in ②,then cultured in 2.5mL of LB Kanamycin(+) liquid culture medium for 16 hours.


August 5th



Plasmid DNAs were isolated from E.coli cultured in 4 of LB liquid culture medium which we made on 8/4 by the alkaline-lysis method, we added the DNA to 20uL TE and added 10uL of the solution to 2uL of 6×Dye. Then we electrophoresed it with marker, 2uL of pDONR+ 2uL of 6×Dye+ 8uL of TE,in 0.7% agarose gel in following order (from left to right)

Left control pDONR DNA, BP TNFA-1,-2,-3,-4

Results



August 6th



E.coli carrying the plasmid sample -1 and -2 in LB Kanamycin(+)liquid culture medium for 16 hours.


August 7th



1. Purification of candidate pENTR-TNFAIP3
We purified candidate plasmid-1 and -2 by QIA Prep Spin Miniprep Kit, dissolved it in 30uL of Buffer EB.
Then,the samples were electrophoresed in 0.7% agarose gel.

We made samples as follows.
   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 


We electrophoresed them in following order.
Left marker 5uL control pDONR DNA ,pENTR-TNFAIP3-1 DNA,pENTR-TNFAIP3 -2 DNA marker 4uL marker 3uL Wright.

Results

Concentration of pENTR-TNFAIP3-2 DNA was estimated at around 100ng/uL.


2. LR reaction
We made following solution(vials)in 1.5mL tube.
 BP TNFA-2  2μL 
 pTFW(Destination vector)  0.5μL 
 TE buffer  6.5μL 
 total  9μL 

we added 1uL of LR clonaseⅡ enzyme mix to this solution and incubate it for 2.5 hours.

3. Transformation of E.coli with LR reaction products.
Transformation of E.coli was carried out by adding 2uL of LR reaction products to 100uL of XL1-Blue and plated on the LB ampicillin(+)plate and cultured at 37°C.