Team:KIT-Kyoto/Notebook-week1p

From 2012.igem.org

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<td width="800px" height="510px" valign="top">
<td width="800px" height="510px" valign="top">
<div id="MIGI">
<div id="MIGI">
 +
 +
<h2>First</h2>
 +
<br>
 +
The first number 1 indicates experiments about UAS.
 +
<br>
 +
The first number 2 indicates experiments about GAL4.
 +
<br>
 +
Second number 1 indicates making two experiments at the same time when we carried out ligation.
 +
<br>
 +
Second number 2 indicates making three experiments at the same time when we carried out ligation.
 +
<br>
 +
The parts we are going to submit are these seven, pSB1C3-UAS, pSB1C3-UAS-LacZ, pSB1C3-UAS-EGFP, pSB1C3-UAS-TNFAIP3,pSB1C3-GAL4, pSB1C3-HS-GAL4, and pSB1C3-Act5c-GAL4.
 +
<br><br>
 +
<h2>August 23rd</h2>
<h2>August 23rd</h2>
<br>
<br>
-
Transformation of E. coli with pEGFP-C2 DNA and pAct5C-GAL4 DNA
+
<strong>1-1-1 and 2-1-1 Transformation of E. coli with pEGFP-C2 DNA and pAct5C-GAL4 DNA</strong>
<br>
<br>
E. coli transformed with pEGFP-C2 was spread on the LB Kanamycin(+) plate and that transformed with pAct5C GAL4 was spread on the LB ampicillin(+) plate.
E. coli transformed with pEGFP-C2 was spread on the LB Kanamycin(+) plate and that transformed with pAct5C GAL4 was spread on the LB ampicillin(+) plate.
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<h2>August 24th</h2>
<h2>August 24th</h2>
 +
<br>
 +
<strong>1-1-2 and 2-1-2</strong>
<br>
<br>
The appropriate colonies were picked up and cultured in the LB medium containing the appropriate antibiotics.
The appropriate colonies were picked up and cultured in the LB medium containing the appropriate antibiotics.
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<h2>August 25th</h2>
<h2>August 25th</h2>
<br>
<br>
-
1. Purification of the pEGFP-C2 DNA and the pAct5C-GAL4 DNA
+
<strong>1-1-3 and 2-1-3 Purification of the pEGFP-C2 DNA and the pAct5C-GAL4 DNA</strong>
<br>
<br>
These plasmid DNAs were purified from E. coli by QIA prep Spin Miniprep Kit.
These plasmid DNAs were purified from E. coli by QIA prep Spin Miniprep Kit.
<br><br>
<br><br>
-
2. Digestion of pEGFP-C2 DNA with BamHⅠ and BglⅡt
+
<strong>1-1-4 Digestion of pEGFP-C2 DNA with BamHⅠ and BglⅡt</strong>
<br>
<br>
Restriction enzyme digestion was carried out in the following reaction
Restriction enzyme digestion was carried out in the following reaction
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</Table>
</Table>
<br><br>
<br><br>
-
 
+
<strong>1-1-5 Agarose gel electrophoresis of the digested DNA </strong>
-
3. Agarose gel electrophoresis of the digested DNA  
+
<br>
<br>
The digested DNA was applied to the agarose gel electrophoresis and linearized DNA was purified by QIA quick Gel Extraction Kit.
The digested DNA was applied to the agarose gel electrophoresis and linearized DNA was purified by QIA quick Gel Extraction Kit.
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<img src="http://2012.igem.org/wiki/images/d/d0/0825kit.png" width="500" height="300">
<img src="http://2012.igem.org/wiki/images/d/d0/0825kit.png" width="500" height="300">
<br><br>
<br><br>
-
4. Digestion of pSB1C3DNA with EcoRⅠ and SpeⅠ
+
<strong>1-1-6 Digestion of pSB1C3DNA with EcoRⅠ and SpeⅠ</strong>
<br>
<br>
pSB1C3 DNA was digested with EcoRⅠ and SpeⅠ at 37℃ for 16 hours.
pSB1C3 DNA was digested with EcoRⅠ and SpeⅠ at 37℃ for 16 hours.
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<br><BR>
<br><BR>
-
5. PCR amplification of the UAS region and pSB1C3 DNA
+
<strong>1-1-7 PCR amplification of the UAS region and pSB1C3 DNA</strong>
<br>
<br>
DNA fragments containing UAS was amplified from the pUAST DNA and the BglⅡ site-containing pSB1C3 was also amplified by PCR.
DNA fragments containing UAS was amplified from the pUAST DNA and the BglⅡ site-containing pSB1C3 was also amplified by PCR.
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</Table>
</Table>
<br><br>
<br><br>
 +
 +
 +
 +
<h2>August 26th</h2>
<h2>August 26th</h2>
<br>
<br>
-
・Purification of the digested pSB1C3  
+
<strong>1-1-8 Purification of the digested pSB1C3</strong>
<br>
<br>
pSB1C3 DNA double-digested with EcoRⅠ and SpeⅠ(prepared on 8/25) was applied to the agarose gel electrophoresis. The DNA fragment of interest was purified from the gel by QIA quick Gel Extraction Kit.
pSB1C3 DNA double-digested with EcoRⅠ and SpeⅠ(prepared on 8/25) was applied to the agarose gel electrophoresis. The DNA fragment of interest was purified from the gel by QIA quick Gel Extraction Kit.
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<h2>August 27th</h2>
<h2>August 27th</h2>
<br>
<br>
-
1. Removing the multi-cloning site of the pEGFP-C2
+
<strong>1-1-9 Removing the multi-cloning site of the pEGFP-C2</strong>
<br>
<br>
pEGFP-C2 DNA digested with BglⅡ and BamHⅠwas self ligated in the following reaction.
pEGFP-C2 DNA digested with BglⅡ and BamHⅠwas self ligated in the following reaction.
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</Table>
</Table>
<br><BR>
<br><BR>
-
2. The ligated products were transformed into the E. coli XL-1 blue and the E. coli was apreaded on the LB Kanamycin(+) plate and cultured at 37℃ for 16 hours.
+
<strong>1-1-10</strong>
 +
<br>
 +
The ligated products were transformed into the E. coli XL-1 blue and the E. coli was apreaded on the LB Kanamycin(+) plate and cultured at 37℃ for 16 hours.
<br><br>
<br><br>
-
3. Agarose gel electrophoresis of the DNA fragments containing UAS and the PSR products from the pSB1C3  
+
<strong>1-1-11 Agarose gel electrophoresis of the DNA fragments containing UAS and the PSR products from the pSB1C3 </strong>
<br>
<br>
PCRproducts prepared on August 25th were applied to the agarose gel electrophoresis as shown below.
PCRproducts prepared on August 25th were applied to the agarose gel electrophoresis as shown below.
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<img src="http://2012.igem.org/wiki/images/9/99/0826ckit.png" width="500" height="300">
<img src="http://2012.igem.org/wiki/images/9/99/0826ckit.png" width="500" height="300">
<br><br>
<br><br>
 +
 +
 +
<h2>August 28th</h2>
<h2>August 28th</h2>
<br>
<br>
-
1. Single colony isolation from the E. coli transformed with pEGFP-C2 DNA
+
<strong>1-1-12 Single colony isolation from the E. coli transformed with pEGFP-C2 DNA</strong>
<br>
<br>
Single colony isolationwas carried out from the plate prepared on 8/27and cultured in 2.5mL LB Kanamycin(+) medium at 37℃ for 16 hours.
Single colony isolationwas carried out from the plate prepared on 8/27and cultured in 2.5mL LB Kanamycin(+) medium at 37℃ for 16 hours.
<br><br>
<br><br>
-
2. Transformation of E. coli with pGaTB DNA or pAct5C-GAL4 DNA
+
<strong>2-1-4 Transformation of E. coli with pGaTB DNA or pAct5C-GAL4 DNA</strong>
<br>
<br>
E. coli Xl-1 blue was transformed with pGaTBDNA or pAct5C-GAL4 DNA and cultured on LB ampicillin(+) plate for 16 hours at 37℃.
E. coli Xl-1 blue was transformed with pGaTBDNA or pAct5C-GAL4 DNA and cultured on LB ampicillin(+) plate for 16 hours at 37℃.
<br><br>
<br><br>
 +
 +
<h2>August 29th</h2>
<h2>August 29th</h2>
<br>
<br>
-
1. Single colony isolation from the E. coli transformed with pGaTB DNA or pAct5C-GAL4 DNA
+
<strong>2-1-5 Single colony isolation from the E. coli transformed with pGaTB DNA or pAct5C-GAL4 DNA</strong>
<br>
<br>
From the plate prepared on 8/28 single colonies were isolated and cultured in 2.5mL LB ampicillin(+) medium for 16 hours at 37℃.
From the plate prepared on 8/28 single colonies were isolated and cultured in 2.5mL LB ampicillin(+) medium for 16 hours at 37℃.
<br><br>
<br><br>
-
2. Purification of pEGFP-C2 DNA
+
<strong>1-1-13 Purification of pEGFP-C2 DNA</strong>
<br>
<br>
pEGFP-C2 DNA was purified from E. coli prepared on 8/28 by QIA prep Spin Miniprep Kit.
pEGFP-C2 DNA was purified from E. coli prepared on 8/28 by QIA prep Spin Miniprep Kit.
<br><br>
<br><br>
-
3. Cut check of the pEGFP-C2 DNA
+
<strong>1-1-14 Cut check of the pEGFP-C2 DNA</strong>
<br>
<br>
-
The purified pEGFP-C2 was digested with XhoⅠ and PstⅠ in the following reactions.
+
The purified pEGFP-C2(1-1-13) was digested with XhoⅠ and PstⅠ in the following reactions.
<br><br>
<br><br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
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Results: The DNAs were undigested by these enzymes, confirming that the multi-cloning site of the pEGFP-C2 was successfully removed.
Results: The DNAs were undigested by these enzymes, confirming that the multi-cloning site of the pEGFP-C2 was successfully removed.
<br><br>
<br><br>
-
4. PCR amplification of the DNA fragments containing UAS and EGFP
+
<strong>1-1-15 PCR amplification of the DNA fragments containing UAS and EGFP</strong>
<br>
<br>
Since we found that the previously used primers for PCR were wrong, PCR amplification of the DNA fragments containing UAS and EGFP region were carried out in the following reactions.
Since we found that the previously used primers for PCR were wrong, PCR amplification of the DNA fragments containing UAS and EGFP region were carried out in the following reactions.
<br><br>
<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>

Latest revision as of 13:42, 26 September 2012






First


The first number 1 indicates experiments about UAS.
The first number 2 indicates experiments about GAL4.
Second number 1 indicates making two experiments at the same time when we carried out ligation.
Second number 2 indicates making three experiments at the same time when we carried out ligation.
The parts we are going to submit are these seven, pSB1C3-UAS, pSB1C3-UAS-LacZ, pSB1C3-UAS-EGFP, pSB1C3-UAS-TNFAIP3,pSB1C3-GAL4, pSB1C3-HS-GAL4, and pSB1C3-Act5c-GAL4.

August 23rd


1-1-1 and 2-1-1 Transformation of E. coli with pEGFP-C2 DNA and pAct5C-GAL4 DNA
E. coli transformed with pEGFP-C2 was spread on the LB Kanamycin(+) plate and that transformed with pAct5C GAL4 was spread on the LB ampicillin(+) plate.

August 24th


1-1-2 and 2-1-2
The appropriate colonies were picked up and cultured in the LB medium containing the appropriate antibiotics.

August 25th


1-1-3 and 2-1-3 Purification of the pEGFP-C2 DNA and the pAct5C-GAL4 DNA
These plasmid DNAs were purified from E. coli by QIA prep Spin Miniprep Kit.

1-1-4 Digestion of pEGFP-C2 DNA with BamHⅠ and BglⅡt
Restriction enzyme digestion was carried out in the following reaction

 Total DNA amount  500ng  1ug 
 pEGFP-C  3uL  6uL 
 H buffer(TOYOBO)  5uL  5uL 
 BamHⅠ(TOYOBO)  1uL  1uL 
 BglⅡ(TOYOBO)  1uL  1uL 
 100×BSA  0.5uL  0.5uL 
 dH2O  39.5uL  36.5uL 
 Total  50uL  50uL 


1-1-5 Agarose gel electrophoresis of the digested DNA
The digested DNA was applied to the agarose gel electrophoresis and linearized DNA was purified by QIA quick Gel Extraction Kit.

Photograph of the agarose gel


1-1-6 Digestion of pSB1C3DNA with EcoRⅠ and SpeⅠ
pSB1C3 DNA was digested with EcoRⅠ and SpeⅠ at 37℃ for 16 hours.

 DNA sample  23uL 
 4 buffer(NEB)  5uL 
 EcoRⅠ-HF(NEB)  1uL 
 SpeⅠ(NEB)  1.5uL 
 100×BSA  0.5uL 
 dH2O  19uL 
 Total  50uL 


1-1-7 PCR amplification of the UAS region and pSB1C3 DNA
DNA fragments containing UAS was amplified from the pUAST DNA and the BglⅡ site-containing pSB1C3 was also amplified by PCR.

 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  Circle 
  95℃ 2min
  95℃ 15sec 25 cycle 
  58℃ 30min(UAS) or 2min10sec(pSB1C3) 25 cycle 
  68℃ 2min30sec 25 cycle 
  68℃ 2min30sec
  14℃ ∞


August 26th


1-1-8 Purification of the digested pSB1C3
pSB1C3 DNA double-digested with EcoRⅠ and SpeⅠ(prepared on 8/25) was applied to the agarose gel electrophoresis. The DNA fragment of interest was purified from the gel by QIA quick Gel Extraction Kit.

Photo of the agarose gel


August 27th


1-1-9 Removing the multi-cloning site of the pEGFP-C2
pEGFP-C2 DNA digested with BglⅡ and BamHⅠwas self ligated in the following reaction.

 pEGFP-C2(cut)  1uL 
 Ligation high  2.5uL 
 dH2O  5uL 
 Total  7.5uL 


1-1-10
The ligated products were transformed into the E. coli XL-1 blue and the E. coli was apreaded on the LB Kanamycin(+) plate and cultured at 37℃ for 16 hours.

1-1-11 Agarose gel electrophoresis of the DNA fragments containing UAS and the PSR products from the pSB1C3
PCRproducts prepared on August 25th were applied to the agarose gel electrophoresis as shown below.

Photo of the agarose gel


Since the expected DNA fragments were detected, DNA was purified from the agarose gel by QIA quick Gel Extraction Kit. The purified DNAs were applied to the agarose gel electrophoresis as shown below.

Photo of the agarose gel


August 28th


1-1-12 Single colony isolation from the E. coli transformed with pEGFP-C2 DNA
Single colony isolationwas carried out from the plate prepared on 8/27and cultured in 2.5mL LB Kanamycin(+) medium at 37℃ for 16 hours.

2-1-4 Transformation of E. coli with pGaTB DNA or pAct5C-GAL4 DNA
E. coli Xl-1 blue was transformed with pGaTBDNA or pAct5C-GAL4 DNA and cultured on LB ampicillin(+) plate for 16 hours at 37℃.

August 29th


2-1-5 Single colony isolation from the E. coli transformed with pGaTB DNA or pAct5C-GAL4 DNA
From the plate prepared on 8/28 single colonies were isolated and cultured in 2.5mL LB ampicillin(+) medium for 16 hours at 37℃.

1-1-13 Purification of pEGFP-C2 DNA
pEGFP-C2 DNA was purified from E. coli prepared on 8/28 by QIA prep Spin Miniprep Kit.

1-1-14 Cut check of the pEGFP-C2 DNA
The purified pEGFP-C2(1-1-13) was digested with XhoⅠ and PstⅠ in the following reactions.

 pEGFP-C2-1, -2, -3, -4  1uL 
 10×H buffer(TOYOBO)  0.5uL 
 XhoⅠ  0.1uL 
 PstⅠ  0.1uL 
 dH2O  3.3uL 
 Total  5uL 


The digested samples were applied to the agarose gel electrophoresis.as shown below.

Photo of the agarose gel


Results: The DNAs were undigested by these enzymes, confirming that the multi-cloning site of the pEGFP-C2 was successfully removed.

1-1-15 PCR amplification of the DNA fragments containing UAS and EGFP
Since we found that the previously used primers for PCR were wrong, PCR amplification of the DNA fragments containing UAS and EGFP region were 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(UAS) or 1min(EGFP)  25 cycle 
 68℃  30sec(UAS) or 1min(EGFP) 
 14℃  ∞ 


Agarose gel electrophoresis of the PCR products


Results: DNA fragments containing EGFP were successfully amplified, but not for the UAS.