Team:KIT-Kyoto/Notebook/LabNoteJ/DIAP2-MALT8
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- | {{Template:KIT-Kyoto/ | + | {{Template:KIT-Kyoto/menu1}} |
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{| style="color:#000080;background-color:transparent;" cellpadding="3" cellspacing="1" border="0" bordercolor="#0000FF" width="900px" align="center" | {| style="color:#000080;background-color:transparent;" cellpadding="3" cellspacing="1" border="0" bordercolor="#0000FF" width="900px" align="center" | ||
- | !align="left"|[[Team:KIT-Kyoto | + | !align="left"|[[Team:KIT-Kyoto|Home]] > [[Team:KIT-Kyoto/Notebook|Notebook]] > [[Team:KIT-Kyoto/Notebook/LabNote|Lab Note]] > [[Team:KIT-Kyoto/Notebook/LabNoteJ/DIAP2-MALT8|August]] |
!align="right"|Language:[[Team:KIT-Kyoto/Notebook/LabNote/DIAP2-MALT8|English]]/[[Team:KIT-Kyoto/Notebook/LabNoteJ/DIAP2-MALTJ8|Japanese]] | !align="right"|Language:[[Team:KIT-Kyoto/Notebook/LabNote/DIAP2-MALT8|English]]/[[Team:KIT-Kyoto/Notebook/LabNoteJ/DIAP2-MALTJ8|Japanese]] | ||
|} | |} | ||
- | =='' | + | ==''8th, August''== |
<b>Member</b> | <b>Member</b> | ||
<br> | <br> | ||
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<br> | <br> | ||
- | =='' | + | ==''9th, August''== |
<b>Member</b> | <b>Member</b> | ||
<br> | <br> | ||
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<br> | <br> | ||
- | =='' | + | ==''23rd, August''== |
<b>Member</b> | <b>Member</b> | ||
<br> | <br> | ||
- | : | + | :Matsunami |
<br> | <br> | ||
- | : | + | :Again different annealing conditions were tested. |
:API2-MALT1<br> | :API2-MALT1<br> | ||
Line 301: | Line 301: | ||
:</table> | :</table> | ||
- | : | + | :PCR products were applied to the agarose gel electrophoresis. |
<br> | <br> | ||
<b>Results</b> | <b>Results</b> | ||
- | : | + | :Image of the agarose gel.<BR> |
:<html><body> | :<html><body> | ||
<IMG src="https://static.igem.org/mediawiki/2011/6/68/2011.08.23_API2-MALT1.DIAP2.JPG" width="250px" height="250px" border="0"> | <IMG src="https://static.igem.org/mediawiki/2011/6/68/2011.08.23_API2-MALT1.DIAP2.JPG" width="250px" height="250px" border="0"> | ||
</body></html> | </body></html> | ||
<br> | <br> | ||
- | + | :Lane 1;DNA size marker(1 Kbp ladder) | |
- | + | :Lanes 2 and 3;API2-MALT1 cDNA | |
+ | :Lanes 4 and 5;DIAP2 cDNA | ||
+ | |||
+ | :Again the DNA fragments with the expected sizes were not detected. | ||
+ | |||
<br> | <br> | ||
- | =='' | + | ==''24th, August''== |
<b>Member</b> | <b>Member</b> | ||
<br> | <br> | ||
- | : | + | :Matsunami |
<br> | <br> | ||
- | :API2- | + | :We have decreased the amount of the template API2-MALT1DNA by diluting 10 X and 100X, then used for the PCR reactions under the following conditions. |
:<table border="0"><tr><td> | :<table border="0"><tr><td> | ||
:<table border="0" width="150px"> | :<table border="0" width="150px"> | ||
- | :<tr><td align=center> | + | :<tr><td align=center>PCR reaction</td></tr> |
:</table> | :</table> | ||
:<table border=1 width="250px"> | :<table border=1 width="250px"> | ||
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:</td><TD></TD><TD></TD><td> | :</td><TD></TD><TD></TD><td> | ||
:<table border="0" width="100px"> | :<table border="0" width="100px"> | ||
- | :<tr><td align=center> | + | :<tr><td align=center>Cycle</td></tr> |
:</table> | :</table> | ||
:<table border=1 width="400px"> | :<table border=1 width="400px"> | ||
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:</table> | :</table> | ||
- | : | + | :PCR products were applied to the agarose gel electrophoresis. |
<br> | <br> | ||
<b>Results</b> | <b>Results</b> | ||
- | : | + | :Image of the agarose gel.<BR> |
:<html><body> | :<html><body> | ||
<IMG src="https://static.igem.org/mediawiki/2011/e/e6/2011.08.24_API2-MALT1_10.100%E5%80%8D%E5%B8%8C%E9%87%88.JPG" width="250px" height="250px" border="0"> | <IMG src="https://static.igem.org/mediawiki/2011/e/e6/2011.08.24_API2-MALT1_10.100%E5%80%8D%E5%B8%8C%E9%87%88.JPG" width="250px" height="250px" border="0"> | ||
</body></html> | </body></html> | ||
<br> | <br> | ||
- | + | :Lane 2;DNA size marker(1 Kbp ladder) | |
- | + | :Lanes 3~6;API2-MALT1 cDNA (10 X diluted) | |
+ | :Lanes 8~11;API2-MALT1 cDNA(100 X diluted) | ||
+ | :No amplified DNA was detected. | ||
+ | |||
<br> | <br> | ||
- | =='' | + | ==''25th, August''== |
<b>Member</b> | <b>Member</b> | ||
<br> | <br> | ||
- | : | + | :Matsunami |
<br> | <br> | ||
- | : | + | :Again different annealing conditions were tested. |
:API2-MALT1<br> | :API2-MALT1<br> | ||
Line 378: | Line 385: | ||
:</table> | :</table> | ||
- | : | + | :PCR products were applied to the agarose gel electrophoresis. |
<br> | <br> | ||
<b>Results</b> | <b>Results</b> | ||
- | : | + | :Image of the agarose gel<BR> |
:<html><body> | :<html><body> | ||
<IMG src="https://static.igem.org/mediawiki/2011/7/72/2011.08.25_API2-MALT1.DIAP2.JPG" width="250px" height="250px" border="0"> | <IMG src="https://static.igem.org/mediawiki/2011/7/72/2011.08.25_API2-MALT1.DIAP2.JPG" width="250px" height="250px" border="0"> | ||
</body></html> | </body></html> | ||
<br> | <br> | ||
- | + | :Lane 1;DNA size marker(1 Kbp ladder) | |
- | API2- | + | :Lanes 2 and 3;API2-MALT1 cDNA |
+ | :Lanes 4 and 5;DIAP2 cDNA | ||
+ | :No PCR product was detected for API2-MALT1. The PCR products with the expected size (1.5 kb) were detected for DIAP2. | ||
+ | |||
<br> | <br> | ||
- | =='' | + | ==''29th, August''== |
<b>Member</b> | <b>Member</b> | ||
<br> | <br> | ||
- | : | + | :Takeda、Yokoigawa |
<br> | <br> | ||
- | : | + | :The PCR products for DIAP2 was treated wit phenol-chloroform and then digested with the XhoI for 20 hours at 37°C. |
- | + | ||
:DIAP2<br> | :DIAP2<br> | ||
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:</table> | :</table> | ||
:<table border="1" width="250"> | :<table border="1" width="250"> | ||
- | :<tr><td width="150px" align="center"> | + | :<tr><td width="150px" align="center">PCR reaction in ddH<sub>2</sub>O</td><td width="100px" align="right">44 µl</td></tr> |
:<tr><td align="center">10 x H Buffer</td><td align="right">5 µl</td></tr> | :<tr><td align="center">10 x H Buffer</td><td align="right">5 µl</td></tr> | ||
:<tr><td align="center"><i>Xho</i>Ⅰ</td><td align="right">1 µl</td></tr> | :<tr><td align="center"><i>Xho</i>Ⅰ</td><td align="right">1 µl</td></tr> | ||
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:</table> | :</table> | ||
- | |||
<br> | <br> | ||
- | =='' | + | ==''30th, August''== |
<b>Member</b> | <b>Member</b> | ||
<br> | <br> | ||
- | : | + | :Takeda, Yokoigawa |
<br> | <br> | ||
- | : | + | :The PCR products for DIAP2 was treated wit phenol-chloroform and then digested with the XhoI for 20 hours at 37°C. |
- | + | ||
:<table border="0"><tr><td> | :<table border="0"><tr><td> | ||
Line 439: | Line 446: | ||
:</table> | :</table> | ||
:<table border="1" width="250"> | :<table border="1" width="250"> | ||
- | :<tr><td width="150px" align="center"> | + | :<tr><td width="150px" align="center">PCR reaction in ddH<sub>2</sub>O</td><td width="100px" align="right">44 µl</td></tr> |
:<tr><td align="center">10 x M Buffer</td><td align="right">5 µl</td></tr> | :<tr><td align="center">10 x M Buffer</td><td align="right">5 µl</td></tr> | ||
:<tr><td align="center"><i>Xba</i>Ⅰ</td><td align="right">1 µl</td></tr> | :<tr><td align="center"><i>Xba</i>Ⅰ</td><td align="right">1 µl</td></tr> | ||
Line 456: | Line 463: | ||
:</table></td></tr></table> | :</table></td></tr></table> | ||
- | |||
<br> | <br> | ||
- | =='' | + | ==''31st, August''== |
<b>Member</b> | <b>Member</b> | ||
<br> | <br> | ||
- | : | + | :Takeda, Yokoigawa |
<br> | <br> | ||
- | :[http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickgelextractionkit.aspx QIAquick Gel Extraction Kit] | + | :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]. |
<br> | <br> | ||
<b>Results</b> | <b>Results</b> | ||
- | : | + | :Image of agarose gel after DNA fragment isolation. <br> |
:<html><body> | :<html><body> | ||
<IMG src="https://static.igem.org/mediawiki/2011/2/27/2011.08.31_%E6%A8%AA%E4%BA%95%E5%B7%9DpUAST-flag-vector.JPG" width="240px" height="280px" border="0"> | <IMG src="https://static.igem.org/mediawiki/2011/2/27/2011.08.31_%E6%A8%AA%E4%BA%95%E5%B7%9DpUAST-flag-vector.JPG" width="240px" height="280px" border="0"> | ||
</body></html> | </body></html> | ||
<br> | <br> | ||
- | :pUAST-flag | + | :The amount of the purified pUAST-flag vector was 45.0375 ng/µl. |
- | : | + | :PCR products for DIAP2 Was not successfully recovered from the agarose gel. |
+ | |||
<br> | <br> | ||
</div> | </div> |
Latest revision as of 16:45, 5 October 2011
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8th, August
Member
- Matsunami, Yokoigawa
- To amplify API2-MALT1 cDNA and DIAP2cDNA, PCR reactions were carried out by using the following primers and under the following conditions.
- API2-MALT1
- F primer:GCCGCTCGAGAACATAGTAGAAAACAGCAT
- Tm value 57.8°C
- R primer:GCCGGCTAGCTCATTTTTCAGAAATTCTGA
- Tm value 56.5°C
- amplicon size 3131 bp
- F primer:GCCGCTCGAGAACATAGTAGAAAACAGCAT
- DIAP2
- F primer:GCTTCTCGAGACGGAGCTGGGCATGGAGCT
- Tm value 69°C
- R primer:GCCGTCTAGATCACGAAAGGAACGTGCGCA
- Tm value 66.4°C
- amplicon size 1514 bp
- F primer:GCTTCTCGAGACGGAGCTGGGCATGGAGCT
- 1.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 51.5°C 30sec Extension 72°C 3min20sec +Extension 72°C 10min End 4°C keep
- 2.DIAP2
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 56.9°C 30sec Extension 72°C 1min40sec +Extension 72°C 10min End 4°C keep
9th, August
Member
- Matsunami, Yokoigawa
- 1. We carried out agarose gel electrophoresis to detect the PCR products.
- 2. We transformed E. coli XL-1 blue with the plasmid pUAST-flag.
Results
- 1. Image of the agarose gel.
-
- Lane 1;size marker(1 kbp ladder)
- Lane 2;the amplified API2-MALT1 cDNA fragment
- Lane 3;the amplified DIAP2 cDNA fragment
- Sizes of the fragments were different from the expected sizes.
- 2. The transformed bacterial colonies were detected.
10th, August
Member
- Matsunami
- 1. I have repeated the PCR reactions to amplify API2-MALT1 cDNA and DIAP2 cDNA under the same conditions as those carried out on 8th, August.
- 2. I picked up the colonies and grew them in liquid culture.
Results
- 2. I have successfully grown the transformed bacteria.
11th, August
Member
- Matsunami
- I have isolated the plasmid DNAs (pUAST flag ) by alkaline-lysis method and electrophoresed them in agarose gel.
Results
- Image of the agarose gel.
-
- Lane 1; size marker (1 Kbp ladder)
- Lanes 2~9;pUAST-flag
- Lane 10;The authentic pUAST-flag vector
- Lane 11;the amplified API2-MALT1 cDNA fragment
- Lane 12;the amplified DIAP2 cDNA fragment
12th, August
Member
- Matsunami, Yokoigawa
- 1. Amounts of the isolated plasmid DNA samples were quantified by measuring the absorbance at OD260.
- 2. We grew the transformed bacteria (25 ml x 2) and the plasmid pUAST-flag DNA was isolated by Midi-prep (Invitrogen).
Results
- 1. The absorbance at OD260 of two independent plasmid DNA samples are shown in Table 1.
- Table 1 (OD260)
- sample 1
0.189 0.208 0.192 0.190 0.191 - Average value was 0.194.
- Concentration of DNA was 0.970 µg/µl.
- sample 2
0.282 0.276 0.278 0.269 0.269 - Average value was 0.275.
- Concentration of DNA was 1.37 µg/µl.
2. Image of the agarose gel.
- Lane 1: DNA size marker (1Kb ladder)
- Lane 6: sample 1
- Lane 7: sample 2
- Lane 8: The authentic pUAST-flag vector
- DNAs from samples 1 and 2 migrated to the same positions as the pUAST-flag.
15th, August
Member
- Matsunami, Yokoigawa
- We have tried different Annealing conditions to amplify API2-MALT1 cDNA and DIAP2 cDNA. The other conditions for the PCR reactions were same as those carried out on 8th, August.
- API2-MALT1
Anneling 58.9°C
- DIAP2
Anneling 53°C
- PCR products were applied to the agarose gel electrophoresis.
Results
- Image of the agarose gel.
-
- Lane 1;DNA size marker(1 Kbp ladder)
- Lane 2;API2-MALT1 cDNA
- Lane 3;DIAP2 cDNA
- Size of the amplified fragments for API2-MALT1was different from the expected size and multiple DNA fragments were detected for the DIAP2.
16th, August
Member
- Matsunami
- Again different annealing conditions were tested.
- API2-MALT1
Anneling 53°C
- DIAP2
Anneling 58.9°C
- 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
- Lane 4;DIAP2 cDNA
- In lane 3, the DNA fragment with the expected size (3 kb) with the additional 2kb fragment was detected for API2-MALT1. Multiple :DNA fragments were still detected for the DIAP2.
17th, August
Member
- Matsunami, Yokoigawa
- Again different annealing conditions were tested.
- API2-MALT1
Anneling 53.5°C
- DIAP2
Anneling 50.5°C
- 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
- Lane 4;DIAP2 cDNA
- Size of the amplified fragments for API2-MALT1was different from the expected size and amplified DNA fragments were barely detectable for the DIAP2.
23rd, August
Member
- Matsunami
- Again different annealing conditions were tested.
- API2-MALT1
Anneling 53°C
- DIAP2
Anneling 50.5°C
- 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
- Lanes 4 and 5;DIAP2 cDNA
- Again the DNA fragments with the expected sizes were not detected.
24th, August
Member
- Matsunami
- We have decreased the amount of the template API2-MALT1DNA by diluting 10 X and 100X, then used for the PCR reactions under the following conditions.
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 2;DNA size marker(1 Kbp ladder)
- Lanes 3~6;API2-MALT1 cDNA (10 X diluted)
- Lanes 8~11;API2-MALT1 cDNA(100 X diluted)
- No amplified DNA was detected.
25th, August
Member
- Matsunami
- Again different annealing conditions were tested.
- API2-MALT1
Anneling 51.5°C
- DIAP2
Anneling 50.5°C
- 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
- Lanes 4 and 5;DIAP2 cDNA
- No PCR product was detected for API2-MALT1. The PCR products with the expected size (1.5 kb) were detected for DIAP2.
29th, August
Member
- Takeda、Yokoigawa
- The PCR products for DIAP2 was treated wit phenol-chloroform and then digested with the XhoI for 20 hours at 37°C.
- DIAP2
DIAP2 PCR reaction in ddH2O 44 µl 10 x H Buffer 5 µl XhoⅠ 1 µl total 50 µl pUAST-flag vector pUAST-flag vector(500 ng/µl) 10 µl ddH2O 34 µl 10 x H Buffer 5 µl XhoⅠ 1 µl total 50 µl
30th, August
Member
- Takeda, Yokoigawa
- 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 M Buffer 5 µl XbaⅠ 1 µl total 50 µl pUAST-flag vector pUAST-flag vector(500 ng/µl) 10 μl ddH2O 34 µl 10 x H Buffer 5 µl XhoⅠ 1 µl total 50 µl
31st, August
Member
- Takeda, Yokoigawa
- 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
- Image of agarose gel after DNA fragment isolation.
-
- The amount of the purified pUAST-flag vector was 45.0375 ng/µl.
- PCR products for DIAP2 Was not successfully recovered from the agarose gel.