Team:KIT-Kyoto/Notebook/LabNote/DIAP2-MALT9
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- | + | ==''1st, September''== | |
+ | <b>Member</b> | ||
+ | <br> | ||
+ | :Takeda | ||
+ | <br> | ||
+ | :Again different annealing conditions were tested. | ||
+ | :<table border="0"><tr><td> | ||
+ | :<table border="0" width="150"> | ||
+ | :<tr><td align="center">PCR reaction</td></tr> | ||
+ | :</table> | ||
+ | :<table border="1" width="250"> | ||
+ | :<tr><td width="150" align="center">10 µM Primer F</td><td width="100" align="right">1.5 µl</td></tr> | ||
+ | :<tr><td align="center">10 µM Primer R</td><td align="right">1.5 µl</td></tr> | ||
+ | :<tr><td align="center">Template DNA</td><td align="right">1 µl</td></tr> | ||
+ | :<tr><td align="center">10 x PCR Buffer</td><td align="right">5 µl</td></tr> | ||
+ | :<tr><td align="center">dNTPs</td><td align="right">5 µl</td></tr> | ||
+ | :<tr><td align="center">MgSO<sub>4</sub></td><td align="right">2 µl or 4 µl</td></tr> | ||
+ | :<tr><td align="center">ddH<sub>2</sub>O</td><td align="right">33 µl or 31 µl</td></tr> | ||
+ | :<tr><td align="center">KOD<sup>+</sup> polymelase</td><td align="right">1 µl</td></tr> | ||
+ | :<tr><td> </td><td align="right">total 50 µl</td></tr> | ||
+ | :</table> | ||
+ | :</td><td></td><td></td><td> | ||
+ | :<table border="0" width="100"> | ||
+ | :<tr><td align="center">Cycle</td></tr> | ||
+ | :</table> | ||
+ | :<table border="1" width="400"> | ||
+ | :<tr><td width="100" align="center">Pre-Denature</td><td width="100" align="center">94°C</td><td width="100" align="center">2min</td><td width="100"> </td></tr> | ||
+ | :<tr><td align="center">Denature</td><td align="center">94°C</td><td align="center">15sec</td><td align="center" rowspan="3">35 Cyle</td></tr> | ||
+ | :<tr><td align="center">Anneling</td><td align="center">50.5°C</td><td align="center">30sec</td></tr> | ||
+ | :<tr><td align="center">Extension</td><td align="center">68°C</td><td align="center">100sec</td></tr> | ||
+ | :<tr><td align="center">End</td><td align="center">4°C</td><td align="center">keep</td><td width="100"> </td></tr> | ||
+ | :</table></td></tr></table> | ||
+ | :PCR products were applied to the agarose gel electrophoresis. | ||
+ | <br> | ||
+ | <b>Results</b> | ||
+ | :Image of the agarose gel.<BR> | ||
+ | :<html><body> | ||
+ | <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> | ||
+ | <br> | ||
+ | <br> | ||
+ | ==''5th, September''== | ||
+ | <b>Member</b> | ||
+ | <br> | ||
+ | :Yokoigawa, Takeda | ||
+ | <br> | ||
+ | :The PCR products for DIAP2 was treated wit phenol-chloroform and then digested with the ''Xho''I for 20 hours at 37°C. | ||
+ | :<table border="0"> | ||
+ | :<tr><td>DIAP2</td></tr> | ||
+ | :</table> | ||
+ | :<table border="1" width="250"> | ||
+ | :<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"><i>Xho</i>Ⅰ</td><td align="right">1 μl</td></tr> | ||
+ | :<tr><td> </td><td align="right">total 50 μl</td></tr> | ||
+ | :</table> | ||
+ | <br> | ||
+ | ==''6th, September''== | ||
+ | <b>Member</b> | ||
+ | <br> | ||
+ | :Yokoigawa, Takeda | ||
+ | <br> | ||
+ | :The PCR products for DIAP2 was treated wit phenol-chloroform and then digested with the ''Xba''I for 20 hours at 37°C. | ||
+ | |||
+ | :<table border="0"> | ||
+ | :<tr><td>DIAP2</td></tr> | ||
+ | :</table> | ||
+ | :<table border="1" width="250"> | ||
+ | :<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"><i>Xba</i>Ⅰ</td><td align="right">1 μl</td></tr> | ||
+ | :<tr><td> </td><td align="right">total 50 μl</td></tr> | ||
+ | :</table> | ||
+ | |||
+ | <BR> | ||
+ | |||
+ | ==''7th, September''== | ||
+ | <b>Member</b> | ||
+ | <br> | ||
+ | :Yokoigawa, Takeda | ||
+ | <br> | ||
+ | :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> | ||
+ | <b>Results</b> | ||
+ | :PCR products for DIAP2 was not successfully recovered from the agarose gel. | ||
+ | <br> | ||
+ | |||
+ | ==''12nd, September''== | ||
+ | <b>Member</b> | ||
+ | <br> | ||
+ | :Matsunami, Yokoigawa | ||
+ | <br> | ||
+ | :To amplify DIAP2 cDNA, PCR reactions were carried out by using the following primers and under the following conditions. | ||
+ | |||
+ | :F primer GCTTCTCGATTGACGGAGCTGGGCATGGAGCT<br> | ||
+ | :Tm value 62 °C<br> | ||
+ | :R primer GCCGTCTAGATCACGAAAGGAACGTGCGCA<br> | ||
+ | :Tm value 66.4°C<br> | ||
+ | :amplicon size 1514 bp<br> | ||
+ | <br> | ||
+ | :<table border="0"><tr><td> | ||
+ | :<table border="0" width="150px"> | ||
+ | :<tr><td align=center>PCR reaction</td></tr> | ||
+ | :</table> | ||
+ | :<table border=1 width="250px"> | ||
+ | :<tr><td width="150px" align=center>10 µM Primer F</td><td width="100px" align=right>1.5 µl</td></tr> | ||
+ | :<tr><td align=center>10 µM Primer R</td><td align=right>1.5 µl</td></tr> | ||
+ | :<tr><td align=center>Template DNA</td><td align=right>1 µl</td></tr> | ||
+ | :<tr><td align=center>10 x PCR Buffer</td><td align=right>5 µl</td></tr> | ||
+ | :<tr><td align=center>dNTPs</td><td align=right>5 µl</td></tr> | ||
+ | :<tr><td align=center>MgSO<sub>4</sub></td><td align=right>2 µl</td></tr> | ||
+ | :<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>33 µl</td></tr> | ||
+ | :<tr><td align=center>KOD<sup>+</sup> polymelase</td><td align=right>1 µl</td></tr> | ||
+ | :<tr><td> </td><td align=right>total 50 µl</td></tr> | ||
+ | :</table> | ||
+ | :</td><TD></TD><TD></TD><td> | ||
+ | :<table border="0" width="100px"> | ||
+ | :<tr><td align=center>Cycle</td></tr> | ||
+ | :</table> | ||
+ | :<table border=1 width="400px"> | ||
+ | :<tr><td width="100px" align=center>Pre-Denature</td><td width="100px" align=center>94°C</td><td width="100px" align=center>2min</td><td width="100px"> </td></tr> | ||
+ | :<tr><td align=center>Denature</td><td align=center>94°C</td><td align=center>15sec</td><td align=center rowspan=3>35 Cycle</td></tr> | ||
+ | :<tr><td align=center>Anneling</td><td align=center>50.5°C</td><td align=center>30sec</td></tr> | ||
+ | :<tr><td align=center>Extension</td><td align=center>68°C</td><td align=center>1min40sec</td></tr> | ||
+ | :<tr><td align=center>End</td><td align=center>4°C</td><td align=center>keep</td><td width="100px"> </td></tr> | ||
+ | :</table> | ||
+ | :</td></tr> | ||
+ | :</table> | ||
+ | <br> | ||
+ | |||
+ | :Again different annealing conditions were tested. | ||
+ | : | ||
+ | :Annealing 50.5°C | ||
+ | <BR> | ||
+ | |||
+ | ==''13rd, September''== | ||
+ | <b>Member</b> | ||
+ | <br> | ||
+ | :Matsunami, Yokoigawa | ||
+ | <br> | ||
+ | :PCR products on 12th September were applied to the agarose gel electrophoresis. | ||
+ | <br> | ||
+ | <b>Results</b> | ||
+ | :Image of the agarose gel.<br> | ||
+ | :<html><body> | ||
+ | <IMG src="https://static.igem.org/mediawiki/2011/f/f4/2011.09.13_DIAP2.JPG" width="250px" height="250px" border="0"> | ||
+ | </body></html> | ||
+ | <br> | ||
+ | :Lane 1;DNA size marker(1 Kbp ladder) | ||
+ | :Lanes 2 and 3;DIAP2 cDNA | ||
+ | :Amplified DNA fragments were barely detectable for the DIAP2. | ||
+ | |||
+ | <br> | ||
+ | |||
+ | ==''14th, September''== | ||
+ | <b>Members</b> | ||
+ | <br> | ||
+ | :Matsunami,Yokoigawa | ||
+ | <br> | ||
+ | :To amplify API2-MALT1 cDNA and, PCR reactions were carried out by using the following primers and under the following conditions. | ||
+ | |||
+ | :F primer GCCGCTCGAGACATAGTAGAAAACAGCAT<br> | ||
+ | :Tm value 57.7 °C<br> | ||
+ | :R primer GCCGGCTAGCTCATTTTTCAGAAATTCTGA<br> | ||
+ | :Tm value 56.5 °C<br> | ||
+ | :amplicon size 3131 bp<br> | ||
+ | <br> | ||
+ | :<table border="0"><tr><td> | ||
+ | :<table border="0" width="150px"> | ||
+ | :<tr><td align=center>PCR reaction</td></tr> | ||
+ | :</table> | ||
+ | :<table border=1 width="250px"> | ||
+ | :<tr><td width="150px" align=center>10 µM Primer F</td><td width="100px" align=right>0.4 µl</td></tr> | ||
+ | :<tr><td align=center>10 µM Primer R</td><td align=right>0.4 µl</td></tr> | ||
+ | :<tr><td align=center>Template DNA</td><td align=right>1 µl</td></tr> | ||
+ | :<tr><td align=center>10 x Ex Taq Buffer</td><td align=right>2 µl</td></tr> | ||
+ | :<tr><td align=center>Takara Ex Taq</td><td align=right>0.1 µl</td></tr> | ||
+ | :<tr><td align=center>2.0 mM dNTPs</td><td align=right>2 µl</td></tr> | ||
+ | :<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>14.1 µl</td></tr> | ||
+ | :<tr><td> </td><td align=right>total 20 µl</td></tr> | ||
+ | :</table> | ||
+ | :</td><TD></TD><TD></TD><td> | ||
+ | :<table border="0" width="100px"> | ||
+ | :<tr><td align=center>Cycle</td></tr> | ||
+ | :</table> | ||
+ | :<table border=1 width="400px"> | ||
+ | :<tr><td width="100px" align=center>Pre-Denature</td><td width="100px" align=center>94°C</td><td width="100px" align=center>2min</td><td width="100px"> </td></tr> | ||
+ | :<tr><td align=center>Denature</td><td align=center>94°C</td><td align=center>30sec</td><td align=center rowspan=3>37 Cycle</td></tr> | ||
+ | :<tr><td align=center>Anneling</td><td align=center>51.5°C</td><td align=center>30sec</td></tr> | ||
+ | :<tr><td align=center>Extension</td><td align=center>72°C</td><td align=center>3min20sec</td></tr> | ||
+ | :<tr><td align=center>+Extension</td><td align=center>72°C</td><td align=center>10min</td><td width="100px"> </td></tr> | ||
+ | :<tr><td align=center>End</td><td align=center>4°C</td><td align=center>keep</td><td width="100px"> </td></tr> | ||
+ | :</table> | ||
+ | :</td></tr> | ||
+ | :</table> | ||
+ | |||
+ | :PCR products were applied to the agarose gel electrophoresis. | ||
+ | <br> | ||
+ | <b>Results</b> | ||
+ | :Image of the agarose gel.<br> | ||
+ | :<html><body> | ||
+ | <IMG src="https://static.igem.org/mediawiki/2011/e/ed/%E6%9D%BE%E6%B5%AA%EF%BC%92.JPG" width="250px" height="250px" border="0"> | ||
+ | </body></html> | ||
+ | <br> | ||
+ | :Lane 1;DNA size marker(1 Kbp ladder) | ||
+ | :Lanes 2 and 3;API2-MALT1 cDNA | ||
+ | :No PCR product was detected for API2-MALT1. | ||
+ | <br> | ||
+ | |||
+ | ==''15th, September''== | ||
+ | <b>Members</b> | ||
+ | <br> | ||
+ | :Matsunami,Yokoigawa | ||
+ | <br> | ||
+ | :To amplify API2-MALT1 cDNA and DIAP2 cDNA, PCR reactions were carried out by using under the following conditions. | ||
+ | |||
+ | :API2-MALT1<br> | ||
+ | :<table border="0"><tr><td> | ||
+ | :<table border="0" width="150px"> | ||
+ | :<tr><td align=center>PCR reaction</td></tr> | ||
+ | :</table> | ||
+ | :<table border=1 width="250px"> | ||
+ | :<tr><td width="150px" align=center>10 µM Primer F</td><td width="100px" align=right>0.4 µl</td></tr> | ||
+ | :<tr><td align=center>10 µM Primer R</td><td align=right>0.4 µl</td></tr> | ||
+ | :<tr><td align=center>Template DNA</td><td align=right>1 µl</td></tr> | ||
+ | :<tr><td align=center>10 x Ex Taq Buffer</td><td align=right>2 µl</td></tr> | ||
+ | :<tr><td align=center>Takara Ex Taq</td><td align=right>0.1 µl</td></tr> | ||
+ | :<tr><td align=center>2.0 mM dNTPs</td><td align=right>2 µl</td></tr> | ||
+ | :<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>14.1 µl</td></tr> | ||
+ | :<tr><td> </td><td align=right>total 20 µl</td></tr> | ||
+ | :</table> | ||
+ | :</td><TD></TD><TD></TD><td> | ||
+ | :<table border="0" width="100px"> | ||
+ | :<tr><td align=center>Cycle</td></tr> | ||
+ | :</table> | ||
+ | :<table border=1 width="400px"> | ||
+ | :<tr><td width="100px" align=center>Pre-Denature</td><td width="100px" align=center>94°C</td><td width="100px" align=center>2min</td><td width="100px"> </td></tr> | ||
+ | :<tr><td align=center>Denature</td><td align=center>94°C</td><td align=center>30sec</td><td align=center rowspan=3>37 Cycle</td></tr> | ||
+ | :<tr><td align=center>Anneling</td><td align=center>53°C</td><td align=center>30sec</td></tr> | ||
+ | :<tr><td align=center>Extension</td><td align=center>72°C</td><td align=center>3min20sec</td></tr> | ||
+ | :<tr><td align=center>+Extension</td><td align=center>72°C</td><td align=center>10min</td><td width="100px"> </td></tr> | ||
+ | :<tr><td align=center>End</td><td align=center>4°C</td><td align=center>keep</td><td width="100px"> </td></tr> | ||
+ | :</table> | ||
+ | :</td></tr> | ||
+ | :</table> | ||
+ | <BR> | ||
+ | :DIAP2<br> | ||
+ | :<table border="0"><tr><td> | ||
+ | :<table border="0" width="150px"> | ||
+ | :<tr><td align=center>PCR reaction</td></tr> | ||
+ | :</table> | ||
+ | :<table border=1 width="250px"> | ||
+ | :<tr><td width="150px" align=center>10 µM Primer F</td><td width="100px" align=right>1.5 µl</td></tr> | ||
+ | :<tr><td align=center>10 µM Primer R</td><td align=right>1.5 µl</td></tr> | ||
+ | :<tr><td align=center>Template DNA</td><td align=right>1 µl</td></tr> | ||
+ | :<tr><td align=center>10 x PCR Buffer for KOD Plus</td><td align=right>5 µl</td></tr> | ||
+ | :<tr><td align=center>dNTPs</td><td align=right>5 µl</td></tr> | ||
+ | :<tr><td align=center>MgSO<sub>4</sub></td><td align=right>2 µl</td></tr> | ||
+ | :<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>33 µl</td></tr> | ||
+ | :<tr><td align=center>KOD Plus</td><td align=right>1 µl</td></tr> | ||
+ | :<tr><td> </td><td align=right>total 50 µl</td></tr> | ||
+ | :</table> | ||
+ | :</td><TD></TD><TD></TD><td> | ||
+ | :<table border="0" width="100px"> | ||
+ | :<tr><td align=center>Cycle</td></tr> | ||
+ | :</table> | ||
+ | :<table border=1 width="400px"> | ||
+ | :<tr><td width="100px" align=center>Pre-Denature</td><td width="100px" align=center>94°C</td><td width="100px" align=center>2min</td><td width="100px"> </td></tr> | ||
+ | :<tr><td align=center>Denature</td><td align=center>94°C</td><td align=center>15sec</td><td align=center rowspan=3>35 Cycle</td></tr> | ||
+ | :<tr><td align=center>Anneling</td><td align=center>50.5°C</td><td align=center>30sec</td></tr> | ||
+ | :<tr><td align=center>Extension</td><td align=center>68°C</td><td align=center>1min 40sec</td></tr> | ||
+ | :<tr><td align=center>End</td><td align=center>4°C</td><td align=center>keep</td><td width="100px"> </td></tr> | ||
+ | :</table> | ||
+ | :</td></tr> | ||
+ | :</table> | ||
+ | |||
+ | :PCR products were applied to the agarose gel electrophoresis. | ||
+ | <br> | ||
+ | :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"> | ||
+ | :<tr><td>DIAP2</td></tr> | ||
+ | :</table> | ||
+ | :<table border=1 width="250px"> | ||
+ | :<tr><td width="150px" align=center>PCR products 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><I>Xho</I>Ⅰ</td><td align=right>1 µl</td></tr> | ||
+ | :<tr><td> </td><td align=right>total 50 µl</td></tr> | ||
+ | :</table> | ||
+ | |||
+ | <br> | ||
+ | <b>Results</b> | ||
+ | :Image of the agarose gel.<br> | ||
+ | :<html><body> | ||
+ | <IMG src="https://static.igem.org/mediawiki/2011/8/86/2011.09.15_%E6%9D%BE%E6%B5%AA.JPG" width="250px" height="250px" border="0"> | ||
+ | </body></html> | ||
+ | <br> | ||
+ | :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 | ||
+ | <br> | ||
+ | |||
+ | ==''16th, September''== | ||
+ | <b>Members</b> | ||
+ | <br> | ||
+ | :Matsunami,Yokoigawa | ||
+ | <br> | ||
+ | :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"> | ||
+ | :<tr><td>DIAP2</td></tr> | ||
+ | :</table> | ||
+ | :<table border=1 width="250px"> | ||
+ | :<tr><td width="150px" align=center>PCR products 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><I>Xba</I>Ⅰ</td><td align=right>1 µl</td></tr> | ||
+ | :<tr><td> </td><td align=right>total 50 µl</td></tr> | ||
+ | :</table> | ||
+ | <br> | ||
+ | |||
+ | ==''17th, September''== | ||
+ | <b>Members</b> | ||
+ | <br> | ||
+ | :Matsunami,Yokoigawa | ||
+ | <br> | ||
+ | :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]. | ||
+ | <br> | ||
+ | <b>Results</b> | ||
+ | :Image of agarose gel.<br> | ||
+ | :<html><body> | ||
+ | <IMG src="https://static.igem.org/mediawiki/2011/e/e9/%E6%9D%BE%E6%B5%AA%EF%BC%91.JPG" width="250px" height="250px" border="0"> | ||
+ | </body></html> | ||
+ | <br> | ||
+ | :PCR products for DIAP2 was not successfully recovered from the agarose gel. | ||
+ | <br> | ||
+ | |||
+ | ==''18th, September''== | ||
+ | <b>Member</b> | ||
+ | <br> | ||
+ | :Matsunami | ||
+ | <br> | ||
+ | :I have streaked ''E.coli'' ''DH5 alpha'' on LB plate and incubated for 16h. | ||
+ | <br> | ||
+ | |||
+ | ==''19th, September''== | ||
+ | <b>Member</b> | ||
+ | <br> | ||
+ | :Matsunami | ||
+ | <br> | ||
+ | :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. | ||
+ | <br> | ||
+ | |||
+ | |||
+ | |||
+ | ==''21st, September''== | ||
+ | <b>Member</b> | ||
+ | <br> | ||
+ | :Matsunami | ||
+ | <br> | ||
+ | :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. | ||
+ | :<table border="0"><tr><td> | ||
+ | :<table border="0" width="150px"> | ||
+ | :<tr><td align=center>PCR reaction</td></tr> | ||
+ | :</table> | ||
+ | :<table border=1 width="250px"> | ||
+ | :<tr><td width="150px" align=center>10 µM Primer F</td><td width="100px" align=right>1.5 µl</td></tr> | ||
+ | :<tr><td align=center>10 µM Primer R</td><td align=right>1.5 µl</td></tr> | ||
+ | :<tr><td align=center>Template DNA</td><td align=right>1 µl</td></tr> | ||
+ | :<tr><td align=center>10 x PCR Buffer</td><td align=right>5 µl</td></tr> | ||
+ | :<tr><td align=center>dNTPs</td><td align=right>5 µl</td></tr> | ||
+ | :<tr><td align=center>MgSO<sub>4</sub></td><td align=right>2 µl</td></tr> | ||
+ | :<tr><td align=center>ddH<sub>2</sub>O</td><td align=right>33 µl</td></tr> | ||
+ | :<tr><td align=center>KOD<sup>+</sup> polymelase</td><td align=right>1 µl</td></tr> | ||
+ | :<tr><td> </td><td align=right>total 50 µl</td></tr> | ||
+ | :</table> | ||
+ | :</td><TD></TD><TD></TD><td> | ||
+ | :<table border="0" width="100px"> | ||
+ | :<tr><td align=center>Cycle</td></tr> | ||
+ | :</table> | ||
+ | :<table border=1 width="400px"> | ||
+ | :<tr><td width="100px" align=center>Pre-Denature</td><td width="100px" align=center>94°C</td><td width="100px" align=center>2min</td><td width="100px"> </td></tr> | ||
+ | :<tr><td align=center>Denature</td><td align=center>94°C</td><td align=center>15sec</td><td align=center rowspan=3>35 Cycle</td></tr> | ||
+ | :<tr><td align=center>Anneling</td><td align=center>50.5°C</td><td align=center>30sec</td></tr> | ||
+ | :<tr><td align=center>Extension</td><td align=center>68°C</td><td align=center>1kb/min</td></tr> | ||
+ | :<tr><td align=center>End</td><td align=center>4°C</td><td align=center>keep</td><td width="100px"> </td></tr> | ||
+ | :</table> | ||
+ | :</td></tr> | ||
+ | :</table> | ||
+ | |||
+ | :Amplified DNA fragments were detectable for the DIAP2. | ||
+ | <br> | ||
+ | :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"> | ||
+ | <tr><td>DIAP2</td></tr> | ||
+ | </table> | ||
+ | <table border=1 width="250px"> | ||
+ | <tr><td width="150px" align=center>PCR products 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><I>Xho</I>Ⅰ</td><td align=right>1 µl</td></tr> | ||
+ | <tr><td> </td><td align=right>total 50 µl</td></tr> | ||
+ | </table> | ||
+ | <br> | ||
+ | <b>Results</b> | ||
+ | :1. The absorbance OD600 was over at 0.9. | ||
+ | |||
+ | :2.Image of agarose gel.<br> | ||
+ | :<html><body> | ||
+ | <IMG src="https://static.igem.org/mediawiki/2011/4/44/2011.09.21_DIAP2.2-7.JPG" width="250px" height="250px" border="0"> | ||
+ | </body></html> | ||
+ | <br> | ||
+ | :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. | ||
+ | <br> | ||
+ | |||
+ | ==''22nd, September''== | ||
+ | <b>Member</b> | ||
+ | <br> | ||
+ | :Matsunami | ||
+ | <br> | ||
+ | :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"> | ||
+ | :<tr><td>DIAP2</td></tr> | ||
+ | :</table> | ||
+ | :<table border=1 width="250px"> | ||
+ | :<tr><td width="150px" align=center>PCR products 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><I>Xba</I>Ⅰ</td><td align=right>1 µl</td></tr> | ||
+ | :<tr><td> </td><td align=right>total 50 µl</td></tr> | ||
+ | :</table> | ||
+ | <br> | ||
+ | |||
+ | ==''23rd, September''== | ||
+ | <b>Member</b> | ||
+ | <br> | ||
+ | :Matsunami | ||
+ | <br> | ||
+ | :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]. | ||
+ | <br> | ||
+ | <b>Results</b> | ||
+ | :Image of agarose gel.<br> | ||
+ | :<html><body> | ||
+ | <IMG src="https://static.igem.org/mediawiki/2011/1/1f/%E6%9D%BE%E6%B5%AA%EF%BC%94.JPG" width="250px" height="250px" border="0"> | ||
+ | </body></html> | ||
+ | <br> | ||
+ | :PCR products for DIAP2 was not successfully recovered from the agarose gel.<br> | ||
+ | <br> | ||
+ | |||
+ | ==''25th, September''== | ||
+ | <b>Member</b> | ||
+ | <br> | ||
+ | :Matsunami | ||
+ | <br> | ||
+ | :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. | ||
+ | <br> | ||
+ | |||
+ | |||
+ | |||
+ | ==''27th, September''== | ||
+ | <b>Member</b> | ||
+ | <br> | ||
+ | :Matsunami | ||
+ | <br> | ||
+ | :I have made competent cell ''DH5 alpha''. | ||
+ | <br> | ||
+ | <b>Results</b> | ||
+ | :Competency was very low. | ||
+ | <br> | ||
</div> | </div> |
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.