Team:KIT-Kyoto/Notebook-week2p

From 2012.igem.org

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<br><br>
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<img src="http://2012.igem.org/wiki/images/b/b0/0901akit.png" width="500" height="300">
<img src="http://2012.igem.org/wiki/images/b/b0/0901akit.png" width="500" height="300">
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<br><br>
 
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<Table Border Cellspacing="0">
 
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<Tr><Td></Td><Td> All samples </Td></Tr>
 
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<tr><td> DNA template </td><td> 0.3uL </td></tr>
 
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<Tr><Td> 10× KOD plus buffer </Td><Td> 10uL </Td></Tr>
 
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<tr><td> 2mM dNTPs </td><td> 10uL </td></tr><tr>
 
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<td> 25mM MgSO<sub>4</sub> </td><td> 3.2uL </td></tr>
 
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<td> 10P 5’ primer </td><td> 3uL </td></tr>
 
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<td> 10P 3’ primer </td><td> 3uL </td></tr>
 
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<td> KOD plus </td><td> 2uL </td></tr>
 
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<td> dH<sub>2</sub>O </td><td> 68.5uL </td></tr>
 
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<td> Total <td> 100uL </td></tr>
 
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</Table>
 
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<br><BR>
 
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<Table Border Cellspacing="0">
 
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<Tr><Td> Temperature </Td><Td> Time </Td><Td> Circle </Td></Tr>
 
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<tr><td>  95℃ </td><td> 2min </td><Td></Td></tr>
 
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<Tr><Td>  95℃ </Td><Td> 15sec </Td><Td> 25 cycle </Td></Tr>
 
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<tr><td>  59℃ </td><td> 30sec </td><Td> 25 cycle </Td></tr><tr>
 
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<td>  68℃ </td><td> 1min30sec(Act5c and LacZ) or 2min(GAL4 and U-T) </td><Td> 25 cycle </Td></tr><tr>
 
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<td>  68℃ </td><td> 1min30sec(Act5c and LacZ) or 2min(GAL4 and U-T) </td><Td></Td></tr><tr>
 
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<td>  14℃ </td><td> ∞ </td><Td></Td></tr>
 
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</Table>
 
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<br><br>
 
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<img src="http://2012.igem.org/wiki/images/6/67/0901bkit.png" width="500" height="300">
 
<br><br>
<br><br>
2. Expected bands for pAct5C DNA and pGaTB DNA were detected on the gel, but band for UAS fragment was not.
2. Expected bands for pAct5C DNA and pGaTB DNA were detected on the gel, but band for UAS fragment was not.
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<h2>September 4th</h2>
<h2>September 4th</h2>
<br>
<br>
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Digestin of GAL4 fragments by XbaⅠ and SpeⅠ
+
1. Digestion of GAL4 fragments by XbaⅠ and SpeⅠ
<br>
<br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
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<Tr><Td> 9/3 GAL4 </Td><Td> 40uL </Td></Tr>
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<Tr><Td> GAL4 fragments prepared on 9/3 </Td><Td> 40uL </Td></Tr>
<tr><td> M buffer(TOYOBO) </td><td> 5uL </td></tr>
<tr><td> M buffer(TOYOBO) </td><td> 5uL </td></tr>
<Tr><Td> XbaⅠ(TOYOBO) </Td><Td> 0.5uL </Td></Tr>
<Tr><Td> XbaⅠ(TOYOBO) </Td><Td> 0.5uL </Td></Tr>
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</Table>
</Table>
<br><BR>
<br><BR>
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Digestion of pSB1C3 DNA, LacZ fragments and EGFP fragments with BglⅡ
+
2. Digestion of pSB1C3 DNA, LacZ fragments and EGFP fragments with BglⅡ
<br>
<br>
 +
pSB1C3 DNA prepared on 8/27, LacZ fragment prepared on 9/3 and EGFP fragments were digested with BglⅡ in the following conditions.
 +
<br>
 +
<br>
 +
3. Digestion of pSB1C3 DNA with XbaⅠ and SpeⅠ
 +
<br>
 +
pSB1C3 DNA (vector) prepared on 8/23 was double digested with XbaⅠ and SpeⅠ.
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<br><br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
<Tr><Td> each DNA </Td><Td> 40uL </Td></Tr>
<Tr><Td> each DNA </Td><Td> 40uL </Td></Tr>
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<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
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<Tr><Td> 9/3 GAL4 </Td><Td> 23uL </Td></Tr>
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<Tr><Td> pSB1C3 DNA  </Td><Td> 23uL </Td></Tr>
<tr><td> M buffer(TOYOBO) </td><td> 10uL </td></tr>
<tr><td> M buffer(TOYOBO) </td><td> 10uL </td></tr>
<Tr><Td> XbaⅠ(TOYOBO) </Td><Td> 1uL </Td></Tr>
<Tr><Td> XbaⅠ(TOYOBO) </Td><Td> 1uL </Td></Tr>
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After agarose gel electrophoresis, the digested GAL4 fragments were purified by QIA quick Gel Extraction Kit
After agarose gel electrophoresis, the digested GAL4 fragments were purified by QIA quick Gel Extraction Kit
<br><br>
<br><br>
 +
Photo of agarose gel
 +
<br>
<img src="http://2012.igem.org/wiki/images/b/b0/0904kit.png" width="500" height="300">
<img src="http://2012.igem.org/wiki/images/b/b0/0904kit.png" width="500" height="300">
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Ligation was carried out in the following reactions.
Ligation was carried out in the following reactions.
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<br><br>
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<br>
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<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
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<Tr><Td></Td><Td> 1uL </Td></Tr>
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<Tr><Td> pSB1C3 (XbaⅠand SpeⅠdigested) </Td><Td> 1uL </Td></Tr>
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<tr><td></td><td> 1uL </td></tr>
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<tr><td> GAL4 (XbaⅠ and SpeⅠ digested) </td><td> 1uL </td></tr>
<Tr><Td> dH<sub>2</sub>O </Td><Td> 3uL </Td></Tr>
<Tr><Td> dH<sub>2</sub>O </Td><Td> 3uL </Td></Tr>
<Tr><Td> Ligation high </Td><Td> 2.5uL </Td></Tr>
<Tr><Td> Ligation high </Td><Td> 2.5uL </Td></Tr>

Revision as of 15:18, 25 September 2012






August 31st


1. PCR amplification of DNA fragments containing UAS and Heat Shock promoter
We have carried out the PCR reaction for UAS again. PCR amplification of Heat Shock promoter from pCasPer DNA was also carried out in the following reactions.

 DNA template  0.5uL 
 10× KOD plus buffer  10uL 
 2mM dNTPs  10uL 
 25mM MgSO4  3.2uL 
 10P 5’ primer  3uL 
 10P 3’ primer  3uL 
 KOD plus  2uL 
 dH2O  68.3uL 
 Total  100uL 


Reaction conditions
 Temperature Time Cycle
  95℃  2min 
  95℃  15sec  25 cycle 
  58℃  30sec  25 cycle 
  68℃  30sec  25 cycle 
  68℃  30sec 
  14℃  ∞ 


2. Purification of pAct5C DNA and pGaTB DNA
pAct5C DNA and pGaTB DNA was purified from E. coli prepared on 8/29by QIA prep Spin Miniprep Kit.

September 1st


1. Agarose gel electrophoresis of PCR produsts and the purified pAct5C DNA and pGaTB DNA
Agarose gel image is shown below. Left to right: HS promoter 10uL, UAS fragment 10uL, pAct5C DNA-1, pGaTB DNA-1, pGaTB DNA-2, pAct5C DNA



2. Expected bands for pAct5C DNA and pGaTB DNA were detected on the gel, but band for UAS fragment was not.

September 3rd


1. PCR amplification of DNA fragments containing GAL4, Act5C promoter and LacZ sequence
pGaTBDNA was used as a template to amplify GAL4 sequence. pAct5C-LacZ DNA was used to amplify Act5C promoter-enhancer region and LacZ.

 DNA template  0.2uL 
 10× KOD plus buffer  5uL 
 2mM dNTPs  5uL 
 25mM MgSO4  1.6uL 
 10P 5’ primer  1.5uL 
 10P 3’ primer  1.5uL 
 KOD plus  1u L
 dH2O  34.2uL 
 Total  50uL 


Reaction conditions
 Temperature  Time  Circle 
 95℃  2min 
 95℃  15sec  25 cycle 
 55℃(GAL4) and 58℃(Except for GAL4)  30sec  25 cycle 
 68℃  2min30sec  25 cycle 
 68℃  2min30sec 
 14℃  ∞ 


2. Agarose gel electrophoresis of the PCRproducts
Left to right: 1kb ladder marker 5uL GAL4 fragments 10uL Act5C promoter-enhancer 10uL LacZ sequence



Results: Expected PCR products were all detected on the gel.
These PCR products were purified by High Pure PCR Product Kit (Roche).

September 4th


1. Digestion of GAL4 fragments by XbaⅠ and SpeⅠ
 GAL4 fragments prepared on 9/3  40uL 
 M buffer(TOYOBO)  5uL 
 XbaⅠ(TOYOBO)  0.5uL 
 SpeⅠ(TOYOBO)  0.5uL 
 dH2O  4uL 
 Total  50uL 


2. Digestion of pSB1C3 DNA, LacZ fragments and EGFP fragments with BglⅡ
pSB1C3 DNA prepared on 8/27, LacZ fragment prepared on 9/3 and EGFP fragments were digested with BglⅡ in the following conditions.

3. Digestion of pSB1C3 DNA with XbaⅠ and SpeⅠ
pSB1C3 DNA (vector) prepared on 8/23 was double digested with XbaⅠ and SpeⅠ.

 each DNA  40uL 
 3 buffer(NEB)  5uL 
 BglⅡ(NEB)  0.5uL 
 dH2O  4.5uL 
 Total  50uL 


Digestion of pSB1C3 DNA with XbaⅠ and SpeⅠ pSB1C3 DNA (vector) prepared on 8/23 was double digested with XbaⅠ and SpeⅠ.
 pSB1C3 DNA   23uL 
 M buffer(TOYOBO)  10uL 
 XbaⅠ(TOYOBO)  1uL 
 SpeⅠ(TOYOBO)  1uL 
 CIP  0.5uL 
 dH2O  64.5uL 
 Total  100uL 


4. Purification of GAL4 fragments
After agarose gel electrophoresis, the digested GAL4 fragments were purified by QIA quick Gel Extraction Kit

Photo of agarose gel


5. Purification of BglII-digested LacZ fragments
BglII-digested LacZ fragments were purified by High Pure PCR Product Kit.

September 5th


1. Purification of EGFP fragments and HS promoter fragments
EGFP fragments prepared on 8/29 and HS promoter fragments prepared on 8/31were purified by High Pure PCR Product Kit

2. EGFP fragments and pSB1C3 DNA prepared on 8/27were digested with BglⅡ in the following reaction.

 EGFP  pSB1C3 
 DNA template  40uL  23uL 
 3 buffer  5uL  5uL 
 BglⅡ  0.5uL  0.5uL 
 dH2O  4.5uL  21.5uL 
 Total  50uL  50uL 


3. Purification of pSB1C3DNA digested with XbaⅠ and SpeⅠ
Agarose gel electrophoresis image of the purified sample are shown below.



4. SpeI digestion of the BglⅡ-digested LacZ, EGFP, pSB1C3 DNA
SpeⅠ digestion was carried out in the following reactions.

 DNA template  40uL 
 4 buffer(NEB)  5uL 
 SpeⅠ  0.5uL 
 100×BSA  0.5uL 
 CIP  0.5uL 
 dH2O  4uL 
 Total  50uL 


5. Ligation of pSB1C3 DNA and GAL4 fragment
Ligation was carried out in the following reactions.
 pSB1C3 (XbaⅠand SpeⅠdigested)  1uL 
 GAL4 (XbaⅠ and SpeⅠ digested)  1uL 
 dH2O  3uL 
 Ligation high  2.5uL 
 Total  7.5uL 


6. The ligated products were transformed into E. coli XL-1 Blue and spread on the LB Chloramphenicol(+) plate

7. Purification of pSB1C3, EGFP, LacZfragments double digested with BglⅡ and SpeⅠ
The double digested samples were applied to the agarose gel electrophoresis and the DNA fragments were purified by QIA quick Gel Extraction Kit

Photo of agarose gel