Team:KIT-Kyoto/Notebook-week2p

From 2012.igem.org

(Difference between revisions)
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<h2>August 31st</h2>
<h2>August 31st</h2>
<br>
<br>
-
 
+
1. PCR amplification of DNA fragments containing UAS and Heat Shock promoter
 +
<br>
 +
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.
 +
<br><br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
-
<Tr><Td></Td><Td>　All samples　</Td></Tr>
 
<Tr><Td>　DNA template　</Td><Td>　0.5uL　</Td></Tr>
<Tr><Td>　DNA template　</Td><Td>　0.5uL　</Td></Tr>
<tr><td>　10× KOD plus buffer　</td><td>　10uL　</td></tr>
<tr><td>　10× KOD plus buffer　</td><td>　10uL　</td></tr>
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</Table>
</Table>
<br><BR>
<br><BR>
-
 
+
Reaction conditions
 +
<br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
<Tr><Td>　Temperature</Td><Td>　Time　</Td><Td>Cycle</Td></Tr>
<Tr><Td>　Temperature</Td><Td>　Time　</Td><Td>Cycle</Td></Tr>
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</Table>
</Table>
<br><BR>
<br><BR>
 +
2. Purification of pAct5C DNA and pGaTB DNA
 +
<br>
 +
pAct5C DNA and pGaTB DNA was purified from E. coli prepared on 8/29by QIA prep Spin Miniprep Kit.
 +
<br><br>
<h2>September 1st</h2>
<h2>September 1st</h2>
<br>
<br>
 +
1. Agarose gel electrophoresis of PCR produsts and the purified pAct5C DNA and pGaTB DNA
 +
<br>
 +
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
 +
<br><br>
<img src="https://static.igem.org/mediawiki/2012/b/b0/0901akit.png" width="500" height="300">
<img src="https://static.igem.org/mediawiki/2012/b/b0/0901akit.png" width="500" height="300">
<br><br>
<br><br>
 +
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
<Tr><Td></Td><Td>　All samples　</Td></Tr>
<Tr><Td></Td><Td>　All samples　</Td></Tr>
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<img src="https://static.igem.org/mediawiki/2012/6/67/0901bkit.png" width="500" height="300">
<img src="https://static.igem.org/mediawiki/2012/6/67/0901bkit.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.
 +
<br><br>
<h2>September 3rd</h2>
<h2>September 3rd</h2>
<BR>
<BR>
-
 
+
1. PCR amplification of DNA fragments containing GAL4, Act5C promoter and LacZ sequence
 +
<br>
 +
pGaTBDNA was used as a template to amplify GAL4 sequence. pAct5C-LacZ DNA was used to amplify Act5C promoter-enhancer region and LacZ.
 +
<br><br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
-
<Tr><Td></Td><Td>　All samples　</Td></Tr>
 
<tr><td>　DNA template　</td><td>　0.2uL　</td></tr>
<tr><td>　DNA template　</td><td>　0.2uL　</td></tr>
<Tr><Td>　10× KOD plus buffer　</Td><Td>　5uL　</Td></Tr>
<Tr><Td>　10× KOD plus buffer　</Td><Td>　5uL　</Td></Tr>
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</Table>
</Table>
<br><BR>
<br><BR>
 +
Reaction conditions
 +
<br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
<Tr><Td>　Temperature　</Td><Td>　Time　</Td><Td>　Circle　</Td></Tr>
<Tr><Td>　Temperature　</Td><Td>　Time　</Td><Td>　Circle　</Td></Tr>
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<td>　14℃　</td><td>　∞　</td><Td></Td></tr>
<td>　14℃　</td><td>　∞　</td><Td></Td></tr>
</Table>
</Table>
 +
<br><br>
 +
2. Agarose gel electrophoresis of the PCRproducts
 +
<br>
 +
Left to right: 1kb ladder marker 5uL　GAL4 fragments 10uL　Act5C promoter-enhancer 10uL　LacZ sequence
<br><br>
<br><br>
<img src="https://static.igem.org/mediawiki/2012/c/ca/0903kit.png" width="500" height="300">
<img src="https://static.igem.org/mediawiki/2012/c/ca/0903kit.png" width="500" height="300">
 +
<br><br>
 +
Results: Expected PCR products were all detected on the gel.
 +
<br>
 +
These PCR products were purified by High Pure PCR Product Kit (Roche).
<br><br>
<br><br>
<h2>September 4th</h2>
<h2>September 4th</h2>
 +
<br>
 +
Digestin of GAL4 fragments by XbaⅠ and SpeⅠ
<br>
<br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
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</Table>
</Table>
<br><BR>
<br><BR>
 +
Digestion of pSB1C3 DNA, LacZ fragments and EGFP fragments with BglⅡ
 +
<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>
</Table>
<br><BR>
<br><BR>
 +
Digestion of pSB1C3 DNA with XbaⅠ and SpeⅠ
 +
pSB1C3 DNA (vector) prepared on 8/23 was double digested with XbaⅠ and SpeⅠ.
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
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</Table>
</Table>
<br><BR>
<br><BR>
-
 
+
4. Purification of GAL4 fragments
 +
<br>
 +
After agarose gel electrophoresis, the digested GAL4 fragments were purified by QIA quick Gel Extraction Kit
 +
<br><br>
<img src="https://static.igem.org/mediawiki/2012/b/b0/0904kit.png" width="500" height="300">
<img src="https://static.igem.org/mediawiki/2012/b/b0/0904kit.png" width="500" height="300">
 +
<br><br>
 +
5. Purification of BglII-digested LacZ fragments
 +
<br>
 +
BglII-digested LacZ fragments were purified by High Pure PCR Product Kit.
<br><br>
<br><br>
<h2>September 5th</h2>
<h2>September 5th</h2>
<br>
<br>
 +
1. Purification of EGFP fragments and HS promoter fragments
 +
<br>
 +
EGFP fragments prepared on 8/29 and HS promoter fragments prepared on 8/31were purified by High Pure PCR Product Kit
 +
<br><br>
 +
2. EGFP fragments and pSB1C3 DNA prepared on 8/27were digested with BglⅡ in the following reaction.
 +
<br><br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
<Tr><Td></Td><td>　EGFP　</td><Td>　pSB1C3　</Td></Tr>
<Tr><Td></Td><td>　EGFP　</td><Td>　pSB1C3　</Td></Tr>
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</Table>
</Table>
<br><BR>
<br><BR>
-
 
+
3. Purification of pSB1C3DNA digested with XbaⅠ and SpeⅠ
 +
<br>
 +
Agarose gel electrophoresis image of the purified sample are shown below.
 +
<br><br>
<img src="https://static.igem.org/mediawiki/2012/3/3a/0905akit.png" width="500" height="300">
<img src="https://static.igem.org/mediawiki/2012/3/3a/0905akit.png" width="500" height="300">
 +
<br><br>
 +
4. SpeI digestion of the BglⅡ-digested LacZ, EGFP, pSB1C3 DNA
 +
<br>
 +
SpeⅠ digestion was carried out in the following reactions.
<br><br>
<br><br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
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</Table>
</Table>
<br><BR>
<br><BR>
 +
5. Ligation of pSB1C3 DNA and GAL4 fragment
 +
<br>
 +
Ligation was carried out in the following reactions.
 +
 +
<br><br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
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</Table>
</Table>
<br><BR>
<br><BR>
 +
6. The ligated products were transformed into E. coli XL-1 Blue and spread on the LB Chloramphenicol(＋) plate
 +
<br><br>
 +
7. Purification of pSB1C3, EGFP, LacZfragments double digested with BglⅡ and SpeⅠ
 +
<br>
 +
The double digested samples were applied to the agarose gel electrophoresis and the DNA fragments were purified by QIA quick Gel Extraction Kit
 +
<br><br>
 +
Photo of agarose gel
 +
<br>
<img src="https://static.igem.org/mediawiki/2012/6/63/0905bkit.png" width="500" height="300">
<img src="https://static.igem.org/mediawiki/2012/6/63/0905bkit.png" width="500" height="300">
<br><br>
<br><br>

Revision as of 15:05, 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



　All samples　
　DNA template　　0.3uL　
　10× KOD plus buffer　　10uL　
　2mM dNTPs　　10uL　
　25mM MgSO4　　3.2uL　
　10P 5’ primer　　3uL　
　10P 3’ primer　　3uL　
　KOD plus　　2uL　
　dH2O　　68.5uL　
　Total　　100uL　


　Temperature　　Time　　Circle　
　　95℃　　2min　
　　95℃　　15sec　　25 cycle　
　　59℃　　30sec　　25 cycle　
　　68℃　　1min30sec(Act5c and LacZ) or 2min(GAL4 and U-T)　　25 cycle　
　　68℃　　1min30sec(Act5c and LacZ) or 2min(GAL4 and U-T)　
　　14℃　　∞　




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


Digestin of GAL4 fragments by XbaⅠ and SpeⅠ
　9/3 GAL4　　40uL　
　M buffer(TOYOBO)　　5uL　
　XbaⅠ(TOYOBO)　　0.5uL　
　SpeⅠ(TOYOBO)　　0.5uL　
　dH2O　　4uL　
　Total　　50uL　


Digestion of pSB1C3 DNA, LacZ fragments and EGFP fragments with BglⅡ
　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.
　9/3 GAL4　　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



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.

　1uL　
　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


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