Team:KIT-Kyoto/Notebook/LabNote/DIAP2-MALT9
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<IMG src="https://static.igem.org/mediawiki/2011/d/dd/2011.09.01_%E6%A8%AA%E4%BA%95%E5%B7%9DPCR%E5%BE%8CDIAP2.JPG" width="240px" height="280px" border="0"> | <IMG src="https://static.igem.org/mediawiki/2011/d/dd/2011.09.01_%E6%A8%AA%E4%BA%95%E5%B7%9DPCR%E5%BE%8CDIAP2.JPG" width="240px" height="280px" border="0"> | ||
+ | <br> | ||
+ | Lane 1;DNA size marker(1 Kbp ladder)<br> | ||
+ | Lanes 2;DIAP2 cDNA<br> | ||
+ | Amplified DNA fragments were barely detectable for the DIAP2.<br> | ||
</body></html> | </body></html> | ||
<br> | <br> | ||
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:R primer GCCGGCTAGCTCATTTTTCAGAAATTCTGA<br> | :R primer GCCGGCTAGCTCATTTTTCAGAAATTCTGA<br> | ||
:Tm value 56.5 °C<br> | :Tm value 56.5 °C<br> | ||
- | :amplicon | + | :amplicon size 3131 bp<br> |
<br> | <br> | ||
:<table border="0"><tr><td> | :<table border="0"><tr><td> | ||
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:No PCR product was detected for API2-MALT1. | :No PCR product was detected for API2-MALT1. | ||
<br> | <br> | ||
- | |||
==''15th, September''== | ==''15th, September''== | ||
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:Matsunami,Yokoigawa | :Matsunami,Yokoigawa | ||
<br> | <br> | ||
- | :To amplify API2-MALT1 cDNA and | + | :To amplify API2-MALT1 cDNA and DIAP2 cDNA, PCR reactions were carried out by using under the following conditions. |
:API2-MALT1<br> | :API2-MALT1<br> | ||
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:PCR products were applied to the agarose gel electrophoresis. | :PCR products were applied to the agarose gel electrophoresis. | ||
<br> | <br> | ||
- | :The PCR products for DIAP2 was treated | + | :The PCR products for DIAP2 was treated with phenol-chloroform and then digested with the ''Xho''I for 20 hours at 37°C. |
:<table border="0"> | :<table border="0"> | ||
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:Lanes 2~5;DIAP2 cDNA | :Lanes 2~5;DIAP2 cDNA | ||
:Lane 6~8;API2-MALT1 cDNA | :Lane 6~8;API2-MALT1 cDNA | ||
+ | :Amplified DNA fragments were barely detectable for the DIAP2. | ||
+ | :Size of the amplified fragments for API2-MALT1 was different from the expected size | ||
<br> | <br> | ||
- | |||
- | |||
==''16th, September''== | ==''16th, September''== | ||
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:Matsunami,Yokoigawa | :Matsunami,Yokoigawa | ||
<br> | <br> | ||
- | :The PCR products for DIAP2 was treated with phenol-chloroform and then digested with the | + | :The PCR products for DIAP2 was treated with phenol-chloroform and then digested with the ''Xba''I for 20 hours at 37°C. |
:<table border="0"> | :<table border="0"> | ||
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:</table> | :</table> | ||
<br> | <br> | ||
- | |||
- | |||
==''17th, September''== | ==''17th, September''== | ||
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</body></html> | </body></html> | ||
<br> | <br> | ||
- | :PCR products for DIAP2 | + | :PCR products for DIAP2 was not successfully recovered from the agarose gel. |
<br> | <br> | ||
- | |||
- | |||
==''18th, September''== | ==''18th, September''== | ||
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:Matsunami | :Matsunami | ||
<br> | <br> | ||
- | :I have streaked E.coli DH5 alpha on LB plate and incubated for 16h. | + | :I have streaked ''E.coli'' ''DH5 alpha'' on LB plate and incubated for 16h. |
<br> | <br> | ||
- | |||
- | |||
==''19th, September''== | ==''19th, September''== | ||
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:Amplified DNA fragments were detectable for the DIAP2. | :Amplified DNA fragments were detectable for the DIAP2. | ||
<br> | <br> | ||
- | :The PCR products for DIAP2 was treated with phenol-chloroform and then digested with the | + | :The PCR products for DIAP2 was treated with phenol-chloroform and then digested with the ''Xho''I for 20 hours at 37°C. |
<table border="0"> | <table border="0"> | ||
Line 442: | Line 439: | ||
:Lane 1;DNA size marker(1 Kbp ladder) | :Lane 1;DNA size marker(1 Kbp ladder) | ||
:Lanes 2~7;DIAP2 cDNA | :Lanes 2~7;DIAP2 cDNA | ||
+ | :The PCR products with the expected size (1.5 kb) were detected for DIAP2. | ||
<br> | <br> | ||
Line 449: | Line 447: | ||
:Matsunami | :Matsunami | ||
<br> | <br> | ||
- | :The PCR products for DIAP2 was treated with phenol-chloroform and then digested with the | + | :The PCR products for DIAP2 was treated with phenol-chloroform and then digested with the ''Xba''I for 20 hours at 37°C. |
:<table border="0"> | :<table border="0"> | ||
Line 475: | Line 473: | ||
</body></html> | </body></html> | ||
<br> | <br> | ||
- | :PCR products for DIAP2 | + | :PCR products for DIAP2 was not successfully recovered from the agarose gel.<br> |
<br> | <br> | ||
Line 494: | Line 492: | ||
:Matsunami | :Matsunami | ||
<br> | <br> | ||
- | :I have made competent cell DH5 alpha. | + | :I have made competent cell ''DH5 alpha''. |
<br> | <br> | ||
<b>Results</b> | <b>Results</b> |
Latest revision as of 03:25, 6 October 2011
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1st, September
Member
- Takeda
- Again different annealing conditions were tested.
PCR reaction 10 µM Primer F 1.5 µl 10 µM Primer R 1.5 µl Template DNA 1 µl 10 x PCR Buffer 5 µl dNTPs 5 µl MgSO4 2 µl or 4 µl ddH2O 33 µl or 31 µl KOD+ polymelase 1 µl total 50 µl Cycle Pre-Denature 94°C 2min Denature 94°C 15sec 35 Cyle Anneling 50.5°C 30sec Extension 68°C 100sec End 4°C keep
- PCR products were applied to the agarose gel electrophoresis.
Results
- Image of the agarose gel.
-
Lane 1;DNA size marker(1 Kbp ladder)
Lanes 2;DIAP2 cDNA
Amplified DNA fragments were barely detectable for the DIAP2.
5th, September
Member
- Yokoigawa, Takeda
- The PCR products for DIAP2 was treated wit phenol-chloroform and then digested with the XhoI for 20 hours at 37°C.
DIAP2 PCR reaction in ddH2O 44 μl 10 x H Buffer 5 μl XhoⅠ 1 μl total 50 μl
6th, September
Member
- Yokoigawa, Takeda
- The PCR products for DIAP2 was treated wit phenol-chloroform and then digested with the XbaI for 20 hours at 37°C.
DIAP2 PCR reaction in ddH2O 44 μl 10 x M Buffer 5 μl XbaⅠ 1 μl total 50 μl
7th, September
Member
- Yokoigawa, Takeda
- The restriction enzyme-digested PCR fragments and pUAST-flag DNA were extracted from the agarose gel by [http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickgelextractionkit.aspx QIAquick Gel Extraction Kit].
Results
- PCR products for DIAP2 was not successfully recovered from the agarose gel.
12nd, September
Member
- Matsunami, Yokoigawa
- To amplify DIAP2 cDNA, PCR reactions were carried out by using the following primers and under the following conditions.
- F primer GCTTCTCGATTGACGGAGCTGGGCATGGAGCT
- Tm value 62 °C
- R primer GCCGTCTAGATCACGAAAGGAACGTGCGCA
- Tm value 66.4°C
- amplicon size 1514 bp
PCR reaction 10 µM Primer F 1.5 µl 10 µM Primer R 1.5 µl Template DNA 1 µl 10 x PCR Buffer 5 µl dNTPs 5 µl MgSO4 2 µl ddH2O 33 µl KOD+ polymelase 1 µl total 50 µl Cycle Pre-Denature 94°C 2min Denature 94°C 15sec 35 Cycle Anneling 50.5°C 30sec Extension 68°C 1min40sec End 4°C keep
- Again different annealing conditions were tested.
- Annealing 50.5°C
13rd, September
Member
- Matsunami, Yokoigawa
- PCR products on 12th September were applied to the agarose gel electrophoresis.
Results
- Image of the agarose gel.
-
- Lane 1;DNA size marker(1 Kbp ladder)
- Lanes 2 and 3;DIAP2 cDNA
- Amplified DNA fragments were barely detectable for the DIAP2.
14th, September
Members
- Matsunami,Yokoigawa
- To amplify API2-MALT1 cDNA and, PCR reactions were carried out by using the following primers and under the following conditions.
- F primer GCCGCTCGAGACATAGTAGAAAACAGCAT
- Tm value 57.7 °C
- R primer GCCGGCTAGCTCATTTTTCAGAAATTCTGA
- Tm value 56.5 °C
- amplicon size 3131 bp
PCR reaction 10 µM Primer F 0.4 µl 10 µM Primer R 0.4 µl Template DNA 1 µl 10 x Ex Taq Buffer 2 µl Takara Ex Taq 0.1 µl 2.0 mM dNTPs 2 µl ddH2O 14.1 µl total 20 µl Cycle Pre-Denature 94°C 2min Denature 94°C 30sec 37 Cycle Anneling 51.5°C 30sec Extension 72°C 3min20sec +Extension 72°C 10min End 4°C keep
- PCR products were applied to the agarose gel electrophoresis.
Results
- Image of the agarose gel.
-
- Lane 1;DNA size marker(1 Kbp ladder)
- Lanes 2 and 3;API2-MALT1 cDNA
- No PCR product was detected for API2-MALT1.
15th, September
Members
- Matsunami,Yokoigawa
- To amplify API2-MALT1 cDNA and DIAP2 cDNA, PCR reactions were carried out by using under the following conditions.
- API2-MALT1
PCR reaction 10 µM Primer F 0.4 µl 10 µM Primer R 0.4 µl Template DNA 1 µl 10 x Ex Taq Buffer 2 µl Takara Ex Taq 0.1 µl 2.0 mM dNTPs 2 µl ddH2O 14.1 µl total 20 µl Cycle Pre-Denature 94°C 2min Denature 94°C 30sec 37 Cycle Anneling 53°C 30sec Extension 72°C 3min20sec +Extension 72°C 10min End 4°C keep
- DIAP2
PCR reaction 10 µM Primer F 1.5 µl 10 µM Primer R 1.5 µl Template DNA 1 µl 10 x PCR Buffer for KOD Plus 5 µl dNTPs 5 µl MgSO4 2 µl ddH2O 33 µl KOD Plus 1 µl total 50 µl Cycle Pre-Denature 94°C 2min Denature 94°C 15sec 35 Cycle Anneling 50.5°C 30sec Extension 68°C 1min 40sec End 4°C keep
- PCR products were applied to the agarose gel electrophoresis.
- The PCR products for DIAP2 was treated with phenol-chloroform and then digested with the XhoI for 20 hours at 37°C.
DIAP2 PCR products in ddH2O 44 µl 10 x H Buffer 5 µl XhoⅠ 1 µl total 50 µl
Results
- Image of the agarose gel.
-
- Lane 1;DNA size marker(1 Kbp ladder)
- Lanes 2~5;DIAP2 cDNA
- Lane 6~8;API2-MALT1 cDNA
- Amplified DNA fragments were barely detectable for the DIAP2.
- Size of the amplified fragments for API2-MALT1 was different from the expected size
16th, September
Members
- Matsunami,Yokoigawa
- The PCR products for DIAP2 was treated with phenol-chloroform and then digested with the XbaI for 20 hours at 37°C.
DIAP2 PCR products in ddH2O 44 µl 10 x M Buffer 5 µl XbaⅠ 1 µl total 50 µl
17th, September
Members
- Matsunami,Yokoigawa
- The restriction enzyme-digested PCR fragments were extracted from the agarose gel by [http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickgelextractionkit.aspx|QIAquick Gel Extraction Kit].
Results
- Image of agarose gel.
-
- PCR products for DIAP2 was not successfully recovered from the agarose gel.
18th, September
Member
- Matsunami
- I have streaked E.coli DH5 alpha on LB plate and incubated for 16h.
19th, September
Member
- Matsunami
- I have picked a single colony up, transferred it into 250 ml of SOB medium in a flask.
- I have incubated it at 18°C with shaking.
21st, September
Member
- Matsunami
- 1.SOB medium in a flask was quantified by measuring the absorbance at OD600.
- 2. I have repeated the PCR reactions to amplify DIAP2 cDNA under the same conditions as those carried out on 12th September.
PCR reaction 10 µM Primer F 1.5 µl 10 µM Primer R 1.5 µl Template DNA 1 µl 10 x PCR Buffer 5 µl dNTPs 5 µl MgSO4 2 µl ddH2O 33 µl KOD+ polymelase 1 µl total 50 µl Cycle Pre-Denature 94°C 2min Denature 94°C 15sec 35 Cycle Anneling 50.5°C 30sec Extension 68°C 1kb/min End 4°C keep
- Amplified DNA fragments were detectable for the DIAP2.
- The PCR products for DIAP2 was treated with phenol-chloroform and then digested with the XhoI for 20 hours at 37°C.
DIAP2 |
PCR products in ddH2O | 44 µl |
10 x H Buffer | 5 µl |
XhoⅠ | 1 µl |
total 50 µl |
Results
- 1. The absorbance OD600 was over at 0.9.
- 2.Image of agarose gel.
-
- Lane 1;DNA size marker(1 Kbp ladder)
- Lanes 2~7;DIAP2 cDNA
- The PCR products with the expected size (1.5 kb) were detected for DIAP2.
22nd, September
Member
- Matsunami
- The PCR products for DIAP2 was treated with phenol-chloroform and then digested with the XbaI for 20 hours at 37°C.
DIAP2 PCR products in ddH2O 44 µl 10 x M Buffer 5 µl XbaⅠ 1 µl total 50 µl
23rd, September
Member
- Matsunami
- The restriction enzyme-digested PCR fragments were extracted from the agarose gel by [http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickgelextractionkit.aspx|QIAquick Gel Extraction Kit].
Results
- Image of agarose gel.
-
- PCR products for DIAP2 was not successfully recovered from the agarose gel.
25th, September
Member
- Matsunami
- I have picked a single colony up, transferred it into 250 ml of SOB medium in a flask.
- I have incubated it at 18°C with shaking.
27th, September
Member
- Matsunami
- I have made competent cell DH5 alpha.
Results
- Competency was very low.