Team:KIT-Kyoto/Notebook-week2p

From 2012.igem.org

(Difference between revisions)
 
Line 139: Line 139:
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<strong> 2-1-7 Purification of pAct5C DNA and pGaTB DNA</strong><br>
<strong> 2-1-7 Purification of pAct5C DNA and pGaTB DNA</strong><br>
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<br>
 
pAct5C DNA and pGaTB DNA was purified from E. coli prepared on 8/29by QIA prep Spin Miniprep Kit.
pAct5C DNA and pGaTB DNA was purified from E. coli prepared on 8/29by QIA prep Spin Miniprep Kit.
<br><br>
<br><br>
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Agarose gel electrophoresis of PCR produsts and the purified pAct5C DNA and pGaTB DNA</strong><br>
Agarose gel electrophoresis of PCR produsts and the purified pAct5C DNA and pGaTB DNA</strong><br>
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<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
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>
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<strong>1-1-21 and 2-1-11
<strong>1-1-21 and 2-1-11
PCR amplification of DNA fragments containing GAL4, Act5C promoter and LacZ sequence</strong><br>
PCR amplification of DNA fragments containing GAL4, Act5C promoter and LacZ sequence</strong><br>
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<br>
 
pGaTBDNA was used as a template to amplify GAL4 sequence. pAct5C-LacZ DNA was used to amplify Act5C promoter-enhancer region and LacZ.  
pGaTBDNA was used as a template to amplify GAL4 sequence. pAct5C-LacZ DNA was used to amplify Act5C promoter-enhancer region and LacZ.  
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<strong>1-1-22 and 2-1-12
<strong>1-1-22 and 2-1-12
   Agarose gel electrophoresis of the PCRproducts</strong><br>
   Agarose gel electrophoresis of the PCRproducts</strong><br>
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<br>
 
Left to right: 1kb ladder marker 5uL　GAL4 fragments 10uL　Act5C promoter-enhancer 10uL　LacZ sequence  
Left to right: 1kb ladder marker 5uL　GAL4 fragments 10uL　Act5C promoter-enhancer 10uL　LacZ sequence  
<br><br>
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<strong>1-1-24
<strong>1-1-24
  Digestion of pSB1C3 DNA, LacZ fragments and EGFP fragments with BglⅡ</strong><br>
  Digestion of pSB1C3 DNA, LacZ fragments and EGFP fragments with BglⅡ</strong><br>
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<br>
 
pSB1C3 DNA prepared on 8/27, LacZ fragment prepared on 9/3 and EGFP fragments were digested with BglⅡ in the following conditions.
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>
<br>
<br>
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<strong>1-1-24 Digestion of pSB1C3 DNA with XbaⅠ and SpeⅠ</strong>
+
<strong>1-1-25 Digestion of pSB1C3 DNA with XbaⅠ and SpeⅠ</strong>
<br>
<br>
pSB1C3 DNA (vector) prepared on 8/23 was double digested with XbaⅠ and SpeⅠ.
pSB1C3 DNA (vector) prepared on 8/23 was double digested with XbaⅠ and SpeⅠ.
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<strong>2-1-16
<strong>2-1-16
  Purification of GAL4 fragments</strong><br>
  Purification of GAL4 fragments</strong><br>
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<br>
 
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>
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<strong>1-1-25
<strong>1-1-25
  Purification of BglII-digested LacZ fragments</strong><br>
  Purification of BglII-digested LacZ fragments</strong><br>
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<br>
 
BglII-digested LacZ fragments were purified by High Pure PCR Product Kit.
BglII-digested LacZ fragments were purified by High Pure PCR Product Kit.
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<strong>1-1-26 and 2-1-17
<strong>1-1-26 and 2-1-17
Purification of EGFP fragments and HS promoter fragments</strong><br>
Purification of EGFP fragments and HS promoter fragments</strong><br>
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<br>
 
EGFP fragments prepared on 8/29 and HS promoter fragments prepared on 8/31were purified by High Pure PCR Product Kit
EGFP fragments prepared on 8/29 and HS promoter fragments prepared on 8/31were purified by High Pure PCR Product Kit
<br><br>
<br><br>
<strong>1-1-27
<strong>1-1-27
EGFP fragments and pSB1C3 DNA prepared on 8/27were digested with BglⅡ in the following reaction.</strong><br>
EGFP fragments and pSB1C3 DNA prepared on 8/27were digested with BglⅡ in the following reaction.</strong><br>
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+
<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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Purification of pSB1C3DNA digested with XbaⅠ and SpeⅠ
Purification of pSB1C3DNA digested with XbaⅠ and SpeⅠ
</strong><br>
</strong><br>
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<br>
 
Agarose gel electrophoresis image of the purified sample are shown below.
Agarose gel electrophoresis image of the purified sample are shown below.
<br><br>
<br><br>
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SpeI digestion of the BglⅡ-digested LacZ, EGFP, pSB1C3 DNA</strong><br>
SpeI digestion of the BglⅡ-digested LacZ, EGFP, pSB1C3 DNA</strong><br>
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<br>
 
SpeⅠ digestion was carried out in the following reactions.
SpeⅠ digestion was carried out in the following reactions.
<br><br>
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Ligation of pSB1C3 DNA and GAL4 fragment</strong><br>
Ligation of pSB1C3 DNA and GAL4 fragment</strong><br>
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<br>
 
Ligation was carried out in the following reactions.
Ligation was carried out in the following reactions.
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Purification of pSB1C3, EGFP, LacZfragments double digested with BglⅡ and SpeⅠ
Purification of pSB1C3, EGFP, LacZfragments double digested with BglⅡ and SpeⅠ
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</strong><br><br>
+
</strong><br>
The double digested samples were applied to the agarose gel electrophoresis and the DNA fragments were purified by QIA quick Gel Extraction Kit
The double digested samples were applied to the agarose gel electrophoresis and the DNA fragments were purified by QIA quick Gel Extraction Kit
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<strong>1-1-31 PCR products were applied to the agarose gel electrophoresis</strong><br>
<strong>1-1-31 PCR products were applied to the agarose gel electrophoresis</strong><br>
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<br>
 
From left to right: UAS, UAS-TNFAIP3, pSB1C3
From left to right: UAS, UAS-TNFAIP3, pSB1C3
<br><br>
<br><br>

Latest revision as of 06:44, 26 September 2012

August 31st


1-1-16 and 2-1-6 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℃　　∞　


2-1-7 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


1-1-18 and 2-1-8 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



1-1-19 and 2-1-9 Expected bands for pAct5C DNA and pGaTB DNA were detected on the gel, but band for UAS fragment was not.

September 3rd


1-1-21 and 2-1-11 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℃　　∞　


1-1-22 and 2-1-12 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.

1-1-23 and 2-1-13

These PCR products were purified by High Pure PCR Product Kit (Roche).

September 4th


2-1-14 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　


1-1-24 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.

1-1-25 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　


2-1-15
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　


2-1-16 Purification of GAL4 fragments
After agarose gel electrophoresis, the digested GAL4 fragments were purified by QIA quick Gel Extraction Kit

Photo of agarose gel


1-1-25 Purification of BglII-digested LacZ fragments
BglII-digested LacZ fragments were purified by High Pure PCR Product Kit.

September 5th


1-1-26 and 2-1-17 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

1-1-27 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　


2-1-18 Purification of pSB1C3DNA digested with XbaⅠ and SpeⅠ
Agarose gel electrophoresis image of the purified sample are shown below.



1-1-28 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　


2-1-19 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　


2-1-20
The ligated products were transformed into E. coli XL-1 Blue and spread on the LB Chloramphenicol(＋) plate

1-1-29 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


September 6th



Transformation(2-1-20) performed on September 5th was not successful, since we had no colony on the plate.

1-1-30
PCR amplification of UAS , UAS-TNFAIP3, pSB1C3DNA

Composition
　DNA template　　0.25uL　
　10× KOD plus buffer　　5uL　
　2mM dNTPs　　5uL　
　25mM MgSO4　　1.6uL　
　10P 5’ primer　　1.5uL　
　10P 3’ primer　　1.5uL　
　KOD plus　　1uL　
　dH2O　　34.15uL　
　Total　　50uL　


Reaction conditions
　Temperature　　Time　　Cycle　
　　95℃　　2min　
　　95℃　　15sec　30 cycle　
　　58℃　　30sec　30 cycle　
　　68℃　　30sec(UAS) or 2min10sec(Except for UAS)　　30 cycle　
　　68℃　　30sec(UAS) or 2min10sec(Except for UAS)　
　　14℃　　∞　


1-1-31 PCR products were applied to the agarose gel electrophoresis
From left to right: UAS, UAS-TNFAIP3, pSB1C3

1-1-32 Purifucation of pSB1C3 PCR products
pSB1C3 PCR products were purified from the gel by QIA quick Gel Extraction Kit.



2-1-21 Ligation of pSB1C3 DNA and GAL4 fragment

　pSB1C3(XbaⅠ and SpeⅠ cut)　　1uL　
　GAL4(XbaⅠ and SpeⅠ cut)　　1uL　
　dH2O　　3uL　
　Ligation high　　2.5uL　
　Total　　7.5uL　


2-1-22
The ligation products were transformed into E. coli XL-1and spread on the LB Chloramphenicol(＋) plate

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