Team:KIT-Kyoto/Notebook/LabNote/DIAP2-MALT9

From 2011.igem.org

(Difference between revisions)
(12th, September)
(18th, September)
 
(32 intermediate revisions not shown)
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-
==''9月1日''==
+
==''1st, September''==
<b>Member</b>
<b>Member</b>
<br>
<br>
-
:武田
+
:Takeda
<br>
<br>
-
:DIAP2について、以下の条件でPCRを行った。
+
:Again different annealing conditions were tested. 
:<table border="0"><tr><td>
:<table border="0"><tr><td>
:<table border="0" width="150">
:<table border="0" width="150">
-
:<tr><td align="center">PCR条件</td></tr>
+
:<tr><td align="center">PCR reaction</td></tr>
:</table>
:</table>
:<table border="1" width="250">
:<table border="1" width="250">
Line 34: Line 34:
:</td><td></td><td></td><td>
:</td><td></td><td></td><td>
:<table border="0" width="100">
:<table border="0" width="100">
-
:<tr><td align="center">Cycle条件</td></tr>
+
:<tr><td align="center">Cycle</td></tr>
:</table>
:</table>
:<table border="1" width="400">
:<table border="1" width="400">
Line 44: Line 44:
:</table></td></tr></table>
:</table></td></tr></table>
-
:PCR産物が増幅していることを確認するため、電気泳動を行った。
+
:PCR products were applied to the agarose gel electrophoresis. 
<br>
<br>
<b>Results</b>
<b>Results</b>
-
:泳動後の写真<BR>
+
:Image of the agarose gel.<BR>
:<html><body>
:<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">
<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>
-
:正しい位置にバンドが見られた。
+
 
<br>
<br>
-
==''9月5日''==
+
==''5th, September''==
<b>Member</b>
<b>Member</b>
<br>
<br>
-
:横井川
+
:Yokoigawa, Takeda
<br>
<br>
-
:1日に確認したDIAP2をフェノールクロロホルム処理し、下表に従って制限酵素処理を行った。(37°C、overnight)
+
: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">
:<table border="0">
Line 66: Line 70:
:</table>
:</table>
:<table border="1" width="250">
:<table border="1" width="250">
-
:<tr><td width="150px" align="center">PCR産物 in ddH<sub>2</sub>O</td><td width="100px" align="right">44 μl</td></tr>
+
:<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>
Line 72: Line 76:
:</table>
:</table>
-
:37°Cで20時間静置した
 
<br>
<br>
-
==''9月6日''==
+
==''6th, September''==
<b>Member</b>
<b>Member</b>
<br>
<br>
-
:横井川
+
:Yokoigawa, Takeda
<br>
<br>
-
:1日に確認したDIAP2をフェノールクロロホルム処理し、下表に従って制限酵素処理を行った。(37°C、overnight)
+
: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">
:<table border="0">
Line 86: Line 89:
:</table>
:</table>
:<table border="1" width="250">
:<table border="1" width="250">
-
:<tr><td width="150px" align="center">PCR産物 in ddH<sub>2</sub>O</td><td width="100px" align="right">44 μl</td></tr>
+
:<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 92: Line 95:
:</table>
:</table>
-
:37°Cで20時間静置した。
+
<BR>
-
<br>
+
-
==''9月7日''==
+
==''7th, September''==
<b>Member</b>
<b>Member</b>
<br>
<br>
-
:横井川
+
:Yokoigawa, Takeda
<br>
<br>
-
:[http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickgelextractionkit.aspx QIAquick Gel Extraction Kit]を使用してゲルからDIAP2のDNAを抽出した。
+
: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>
-
:バンドが確認できなかったためゲル抽出を行う事が出来なかった。
+
:PCR products for DIAP2 was not successfully recovered from the agarose gel.
<br>
<br>
-
==''12th, September''==
+
==''12nd, September''==
<b>Member</b>
<b>Member</b>
<br>
<br>
Line 154: Line 156:
<BR>
<BR>
-
==''13th, September''==
+
==''13rd, September''==
<b>Member</b>
<b>Member</b>
<br>
<br>
Line 173: Line 175:
<br>
<br>
-
==''9月14日''==
+
==''14th, September''==
-
<b>Member</b>
+
<b>Members</b>
<br>
<br>
-
:松浪、横井川
+
:Matsunami,Yokoigawa
<br>
<br>
-
:API2-MALT1について以下のプライマーを用いて、以下の条件でPCRを行った。
+
:To amplify API2-MALT1 cDNA and, PCR reactions were carried out by using the following primers and under the following conditions.
-
:F primer GCCGCTCGAGAACATAGTAGAAAACAGCAT<br>
+
:F primer GCCGCTCGAGACATAGTAGAAAACAGCAT<br>
-
:Tm値    57.8 °C<br>  
+
:Tm value    57.7 °C<br>  
:R primer GCCGGCTAGCTCATTTTTCAGAAATTCTGA<br>
:R primer GCCGGCTAGCTCATTTTTCAGAAATTCTGA<br>
-
:Tm値    56.5 °C<br>  
+
:Tm value    56.5 °C<br>  
-
:増幅サイズ  約3131 bp<br>
+
:amplicon size  3131 bp<br>
<br>
<br>
:<table border="0"><tr><td>
:<table border="0"><tr><td>
:<table border="0" width="150px">
:<table border="0" width="150px">
-
:<tr><td align=center>PCR条件</td></tr>
+
:<tr><td align=center>PCR reaction</td></tr>
:</table>
:</table>
:<table border=1 width="250px">
:<table border=1 width="250px">
Line 202: Line 204:
:</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>Cycle条件</td></tr>
+
:<tr><td align=center>Cycle</td></tr>
:</table>
:</table>
:<table border=1 width="400px">
:<table border=1 width="400px">
Line 215: Line 217:
:</table>
:</table>
-
:PCR産物が増幅していることを確認するため、電気泳動を行った。
+
:PCR products were applied to the agarose gel electrophoresis.
<br>
<br>
<b>Results</b>
<b>Results</b>
-
:泳動後の写真<br>
+
:Image of the agarose gel.<br>
:<html><body>
:<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">
<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>
</body></html>
<br>
<br>
-
マーカー(1 Kbp)のみバンドが見られた。
+
:Lane 1;DNA size marker(1 Kbp ladder)
 +
:Lanes 2 and 3;API2-MALT1 cDNA
 +
:No PCR product was detected for API2-MALT1.
<br>
<br>
-
==''9月15日''==
+
==''15th, September''==
-
<b>Member</b>
+
<b>Members</b>
<br>
<br>
-
:松浪、横井川
+
:Matsunami,Yokoigawa
<br>
<br>
-
:API2-MALT1、DIAP2について、以下の条件でPCRを行った。
+
: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>
:<table border="0"><tr><td>
:<table border="0"><tr><td>
:<table border="0" width="150px">
:<table border="0" width="150px">
-
:<tr><td align=center>PCR条件</td></tr>
+
:<tr><td align=center>PCR reaction</td></tr>
:</table>
:</table>
:<table border=1 width="250px">
:<table border=1 width="250px">
Line 250: Line 254:
:</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>Cycle条件</td></tr>
+
:<tr><td align=center>Cycle</td></tr>
:</table>
:</table>
:<table border=1 width="400px">
:<table border=1 width="400px">
Line 266: Line 270:
:<table border="0"><tr><td>
:<table border="0"><tr><td>
:<table border="0" width="150px">
:<table border="0" width="150px">
-
:<tr><td align=center>PCR条件</td></tr>
+
:<tr><td align=center>PCR reaction</td></tr>
:</table>
:</table>
:<table border=1 width="250px">
:<table border=1 width="250px">
Line 281: Line 285:
:</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>Cycle条件</td></tr>
+
:<tr><td align=center>Cycle</td></tr>
:</table>
:</table>
:<table border=1 width="400px">
:<table border=1 width="400px">
Line 293: Line 297:
:</table>
:</table>
-
:PCR産物が増幅していることを確認するため、電気泳動を行った。
+
:PCR products were applied to the agarose gel electrophoresis.
<br>
<br>
-
:DIAP2のPCR産物を、下表に従って制限酵素処理を行った。(37°C、overnight)
+
: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 301: Line 305:
:</table>
:</table>
:<table border=1 width="250px">
:<table border=1 width="250px">
-
:<tr><td width="150px" align=center>PCR産物 in ddH<sub>2</sub>O</td><td width="100px" align=right>44 µl</td></tr>
+
:<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>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>
Line 307: Line 311:
:</table>
:</table>
-
37°Cで20時間静置した。
 
<br>
<br>
<b>Results</b>
<b>Results</b>
-
:泳動後の写真<br>
+
:Image of the agarose gel.<br>
:<html><body>
:<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">
<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>
</body></html>
<br>
<br>
-
:左から順に1レーン;マーカー(1 Kbp)、2~5レーン;DIAP2、6~8レーン;API2-MALT1
+
:Lane 1;DNA size marker(1 Kbp ladder)
-
:DIAP2に関しては予想される位置にバンドが見られたが、API2-MALT1は見られなかった。
+
: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>
<br>
-
==''9月16日''==
+
==''16th, September''==
-
<b>Member</b>
+
<b>Members</b>
<br>
<br>
-
:松浪、横井川
+
:Matsunami,Yokoigawa
<br>
<br>
-
:DIAP2のPCR産物を、下表に従って制限酵素処理を行った。(37°C、overnight)
+
: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 330: Line 336:
:</table>
:</table>
:<table border=1 width="250px">
:<table border=1 width="250px">
-
:<tr><td width="150px" align=center>PCR産物 in ddH<sub>2</sub>O</td><td width="100px" align=right>44 µl</td></tr>
+
:<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>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 337: Line 343:
<br>
<br>
-
==''9月17日''==
+
==''17th, September''==
-
<b>Member</b>
+
<b>Members</b>
<br>
<br>
-
:松浪、横井川
+
:Matsunami,Yokoigawa
<br>
<br>
-
:制限酵素処理を行ったDIAP2のプラスミドを確認するため、電気泳動を行った。
+
: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>
<br>
<b>Results</b>
<b>Results</b>
-
:泳動後の写真<br>
+
:Image of agarose gel.<br>
:<html><body>
:<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">
<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>
</body></html>
<br>
<br>
-
:マーカー(1 Kbp)のバンドのみが見られた。制限酵素処理を行ったDIAP2のサンプルはすべてバンドが見られなかった。
+
:PCR products for DIAP2 was not successfully recovered from the agarose gel.
<br>
<br>
-
==''9月18日''==
+
==''18th, September''==
<b>Member</b>
<b>Member</b>
<br>
<br>
-
:松浪
+
:Matsunami
<br>
<br>
-
:コンピテントセル(DH5α)を作成した。
+
:I have streaked ''E.coli'' ''DH5 alpha'' on LB plate and incubated for 16h.
<br>
<br>
-
==''9月19日''==
+
==''19th, September''==
<b>Member</b>
<b>Member</b>
<br>
<br>
-
:松浪
+
:Matsunami
<br>
<br>
-
:コンピテントセル(DH5α)を作成した。
+
: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>
<br>
-
==''9月21日''==
+
 
 +
 
 +
==''21st, September''==
<b>Member</b>
<b>Member</b>
<br>
<br>
-
:松浪
+
:Matsunami
<br>
<br>
-
:1.コンピテントセル(DH5α)を作成した。
+
:1.SOB medium in a flask was quantified by measuring the absorbance at OD600.
-
 
+
-
:2.DIAP2について、以下の条件でPCRを行った。
+
 +
: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"><tr><td>
:<table border="0" width="150px">
:<table border="0" width="150px">
-
:<tr><td align=center>PCR条件</td></tr>
+
:<tr><td align=center>PCR reaction</td></tr>
:</table>
:</table>
:<table border=1 width="250px">
:<table border=1 width="250px">
Line 395: Line 403:
:</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>Cycle条件</td></tr>
+
:<tr><td align=center>Cycle</td></tr>
:</table>
:</table>
:<table border=1 width="400px">
:<table border=1 width="400px">
Line 407: Line 415:
:</table>
:</table>
-
:PCR産物が増幅していることを確認するため、電気泳動を行った。
+
:Amplified DNA fragments were detectable for the DIAP2.
<br>
<br>
-
:DIAP2のPCR産物を、下表に従って制限酵素処理を行った。(37°C、overnight)
+
: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 415: Line 423:
</table>
</table>
<table border=1 width="250px">
<table border=1 width="250px">
-
<tr><td width="150px" align=center>PCR産物 in ddH<sub>2</sub>O</td><td width="100px" align=right>44 µl</td></tr>
+
<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>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>
Line 422: Line 430:
<br>
<br>
<b>Results</b>
<b>Results</b>
-
:1.ODを測定したが、OD=0.9を超えてしまった。
+
:1. The absorbance OD600 was over at 0.9.
-
:泳動後の写真<br>
+
:2.Image of agarose gel.<br>
:<html><body>
:<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">
<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>
</body></html>
<br>
<br>
-
:左から順に1レーン;マーカー(1 Kbp)、2~7レーン;DIAP2<br>
+
:Lane 1;DNA size marker(1 Kbp ladder)
-
:全てのサンプルでPCRが成功した。
+
:Lanes 2~7;DIAP2 cDNA
 +
:The PCR products with the expected size (1.5 kb) were detected for DIAP2.
<br>
<br>
-
==''9月22日''==
+
==''22nd, September''==
<b>Member</b>
<b>Member</b>
<br>
<br>
-
:不明★
+
:Matsunami
<br>
<br>
-
:DIAP2のPCR産物を、下表に従って制限酵素処理を行った。(37°C、overnight)
+
: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 444: Line 453:
:</table>
:</table>
:<table border=1 width="250px">
:<table border=1 width="250px">
-
:<tr><td width="150px" align=center>PCR産物 in ddH<sub>2</sub>O</td><td width="100px" align=right>44 µl</td></tr>
+
:<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>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 451: Line 460:
<br>
<br>
-
==''9月23日''==
+
==''23rd, September''==
<b>Member</b>
<b>Member</b>
<br>
<br>
-
:松浪
+
:Matsunami
<br>
<br>
-
:制限酵素処理を行ったDIAP2のプラスミドを確認するため、電気泳動を行った。
+
: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>
<br>
<b>Results</b>
<b>Results</b>
-
:泳動後の写真<br>
+
:Image of agarose gel.<br>
:<html><body>
:<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">
<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>
</body></html>
<br>
<br>
-
:マーカー(1 Kbp)のみバンドが見られ、制限酵素処理を行ったDIAP2のサンプルはすべてバンドが見られなった。<br>
+
:PCR products for DIAP2 was not successfully recovered from the agarose gel.<br>
<br>
<br>
-
==''9月25日''==
+
==''25th, September''==
<b>Member</b>
<b>Member</b>
<br>
<br>
-
:松浪
+
:Matsunami
<br>
<br>
-
:コンピテントセル(DH5α)を作成した。
+
: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>
<br>
-
==''9月27日''==
+
 
 +
 
 +
==''27th, September''==
<b>Member</b>
<b>Member</b>
<br>
<br>
-
:松浪
+
:Matsunami
<br>
<br>
-
:コンピテントセル(DH5α)を作成した。
+
:I have made competent cell ''DH5 alpha''.
 +
<br>
 +
<b>Results</b>
 +
:Competency was very low.
<br>
<br>
-
 
</div>
</div>

Latest revision as of 03:25, 6 October 2011



Home Team Project Parts Notebook Safety Human Practice Attributions


Home > Notebook > Lab Note > September Language:English/Japanese

1st, September

Member

Takeda


Again different annealing conditions were tested.
PCR reaction
10 µM Primer F1.5 µl
10 µM Primer R1.5 µl
Template DNA1 µl
10 x PCR Buffer5 µl
dNTPs5 µl
MgSO42 µl or 4 µl
ddH2O33 µl or 31 µl
KOD+ polymelase1 µl
 total 50 µl
Cycle
Pre-Denature94°C2min 
Denature94°C15sec35 Cyle
Anneling50.5°C30sec
Extension68°C100sec
End4°Ckeep 
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 ddH2O44 μl
10 x H Buffer5 μl
Xho1 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 ddH2O44 μl
10 x M Buffer5 μl
Xba1 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 F1.5 µl
10 µM Primer R1.5 µl
Template DNA1 µl
10 x PCR Buffer5 µl
dNTPs5 µl
MgSO42 µl
ddH2O33 µl
KOD+ polymelase1 µl
 total 50 µl
Cycle
Pre-Denature94°C2min 
Denature94°C15sec35 Cycle
Anneling50.5°C30sec
Extension68°C1min40sec
End4°Ckeep 


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 F0.4 µl
10 µM Primer R0.4 µl
Template DNA1 µl
10 x Ex Taq Buffer2 µl
Takara Ex Taq0.1 µl
2.0 mM dNTPs2 µl
ddH2O14.1 µl
 total 20 µl
Cycle
Pre-Denature94°C2min 
Denature94°C30sec37 Cycle
Anneling51.5°C30sec
Extension72°C3min20sec
+Extension72°C10min 
End4°Ckeep 
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 F0.4 µl
10 µM Primer R0.4 µl
Template DNA1 µl
10 x Ex Taq Buffer2 µl
Takara Ex Taq0.1 µl
2.0 mM dNTPs2 µl
ddH2O14.1 µl
 total 20 µl
Cycle
Pre-Denature94°C2min 
Denature94°C30sec37 Cycle
Anneling53°C30sec
Extension72°C3min20sec
+Extension72°C10min 
End4°Ckeep 


DIAP2
PCR reaction
10 µM Primer F1.5 µl
10 µM Primer R1.5 µl
Template DNA1 µl
10 x PCR Buffer for KOD Plus5 µl
dNTPs5 µl
MgSO42 µl
ddH2O33 µl
KOD Plus1 µl
 total 50 µl
Cycle
Pre-Denature94°C2min 
Denature94°C15sec35 Cycle
Anneling50.5°C30sec
Extension68°C1min 40sec
End4°Ckeep 
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 ddH2O44 µl
10 x H Buffer5 µl
Xho1 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 ddH2O44 µl
10 x M Buffer5 µl
Xba1 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 F1.5 µl
10 µM Primer R1.5 µl
Template DNA1 µl
10 x PCR Buffer5 µl
dNTPs5 µl
MgSO42 µl
ddH2O33 µl
KOD+ polymelase1 µl
 total 50 µl
Cycle
Pre-Denature94°C2min 
Denature94°C15sec35 Cycle
Anneling50.5°C30sec
Extension68°C1kb/min
End4°Ckeep 
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 ddH2O44 µl
10 x H Buffer5 µl
Xho1 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 ddH2O44 µl
10 x M Buffer5 µl
Xba1 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.