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| | __NOTOC__ | | __NOTOC__ |
| | {{:Team:KAIST/header}} | | {{:Team:KAIST/header}} |
| - | <table width="100%" border="0"> | + | <table width="100%" border="0" cellpadding = "20px"> |
| | | | |
| | <tr> | | <tr> |
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| | <table width="100%"> | | <table width="100%"> |
| | <tr> | | <tr> |
| - | <td> | + | <td align=center > |
| | <html><a href="https://2010.igem.org/Team:KAIST-Korea/Notebook/Diary/April"><img src="https://static.igem.org/mediawiki/2010/e/e1/April.jpg" width=100></a></html> | | <html><a href="https://2010.igem.org/Team:KAIST-Korea/Notebook/Diary/April"><img src="https://static.igem.org/mediawiki/2010/e/e1/April.jpg" width=100></a></html> |
| | </td> | | </td> |
| - | <td> | + | <td align=center > |
| | <html><a href="https://2010.igem.org/Team:KAIST-Korea/Notebook/Diary/May"><img src="https://static.igem.org/mediawiki/2010/6/6d/5%EC%9B%94.jpg | | <html><a href="https://2010.igem.org/Team:KAIST-Korea/Notebook/Diary/May"><img src="https://static.igem.org/mediawiki/2010/6/6d/5%EC%9B%94.jpg |
| | " width=100></a></html> | | " width=100></a></html> |
| | </td> | | </td> |
| - | <td> | + | <td align=center > |
| | <html><a href="https://2010.igem.org/Team:KAIST-Korea/Notebook/Diary/June"><img src=" https://static.igem.org/mediawiki/2010/3/31/6%EC%9B%94.jpg" width=100></a></html> | | <html><a href="https://2010.igem.org/Team:KAIST-Korea/Notebook/Diary/June"><img src=" https://static.igem.org/mediawiki/2010/3/31/6%EC%9B%94.jpg" width=100></a></html> |
| | | | |
| | </td> | | </td> |
| - | <td> | + | <td align=center > |
| | <html><a href="https://2010.igem.org/Team:KAIST-Korea/Notebook/Diary/July"><img src="https://static.igem.org/mediawiki/2010/4/49/7%EC%9B%94.jpg | | <html><a href="https://2010.igem.org/Team:KAIST-Korea/Notebook/Diary/July"><img src="https://static.igem.org/mediawiki/2010/4/49/7%EC%9B%94.jpg |
| | " width=100></a></html> | | " width=100></a></html> |
| | </td> | | </td> |
| | | | |
| - | <td> | + | <td align=center > |
| | <html><a href="https://2010.igem.org/Team:KAIST-Korea/Notebook/Diary/August"><img src="https://static.igem.org/mediawiki/2010/5/55/8%EC%9B%94.jpg | | <html><a href="https://2010.igem.org/Team:KAIST-Korea/Notebook/Diary/August"><img src="https://static.igem.org/mediawiki/2010/5/55/8%EC%9B%94.jpg |
| | " width=100></a></html> | | " width=100></a></html> |
| | </td> | | </td> |
| - | <td> | + | <td align=center > |
| | <html><a href="https://2010.igem.org/Team:KAIST-Korea/Notebook/Diary/September"><img src="https://static.igem.org/mediawiki/2010/0/02/9%EC%9B%94.jpg | | <html><a href="https://2010.igem.org/Team:KAIST-Korea/Notebook/Diary/September"><img src="https://static.igem.org/mediawiki/2010/0/02/9%EC%9B%94.jpg |
| | " width=100></a></html> | | " width=100></a></html> |
| | </td> | | </td> |
| - | <td> | + | <td align=center > |
| | <html><a href="https://2010.igem.org/Team:KAIST-Korea/Notebook/Diary/October"><img src="https://static.igem.org/mediawiki/2010/9/91/10%EC%9B%94.jpg | | <html><a href="https://2010.igem.org/Team:KAIST-Korea/Notebook/Diary/October"><img src="https://static.igem.org/mediawiki/2010/9/91/10%EC%9B%94.jpg |
| | " width=100></a></html> | | " width=100></a></html> |
| | </td> | | </td> |
| - | <td> | + | <td align=center > |
| | <html><a href="https://2010.igem.org/Team:KAIST-Korea/Notebook/Diary/November"><img src="https://static.igem.org/mediawiki/2010/3/35/11%EC%9B%94.jpg | | <html><a href="https://2010.igem.org/Team:KAIST-Korea/Notebook/Diary/November"><img src="https://static.igem.org/mediawiki/2010/3/35/11%EC%9B%94.jpg |
| | " width=100></a></html> | | " width=100></a></html> |
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| | </tr> | | </tr> |
| | </table> | | </table> |
| | + | |
| | == September 1 == | | == September 1 == |
| | pREP41 vector has arrived. | | pREP41 vector has arrived. |
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| | | | |
| | | | |
| - | ==September ? == | + | ==September 2 ~ September 21 == |
| - | | + | <html> |
| - | | + | We designed PCR schedules and do PCR experiment. When PCR result was not satisfied, then we repeated PCR experiment. There are PCR details on <a href=https://2010.igem.org/Team:KAIST-Korea/Project/Methods/PCR_Result>PCR experiment menu</a>. |
| - | === V_STAT1 (expression) ===
| + | </html> |
| - | | + | |
| - | <center> | + | |
| - | [[Image:PCR_STAT1.jpg]]
| + | |
| - | | + | |
| | <br> | | <br> |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Primer</b></td><td><b>Direction</b></td><td><b>Sequences</b></td><td><b>Length</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>V_STAT_F</td><td>Forward(NdeI)</td><td>5’-<font color=blue>TGTT</font><font color=red>CATATG</font><font color=blue>GCTA</font>ATGTCTCAGTGGTACGAACTTCAG-3’</td><td>24bp</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>V_STAT_R</td><td>Reverse(BamHI)</td><td>5’-<font color=blue>TATC</font><font color=red>GGATCC</font><font color=blue>GAAT</font>TTACACTTCAGACACAGAAATCAAC-3’</td><td>25bp</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| | <br> | | <br> |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Materials</b></td><td><b>Amount</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>STAT1(KRIBB) (template)</td><td>150pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>V_STAT_F</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>V_STAT_R</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>dNTP (dNTP mixture 2.5mM TaKaRa)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)</td><td>0.25ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>10x buffer (10x cloned Pfu Reaction buffer)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>DW (3rd sterile water)</td><td>rest</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Total</td><td>50ul</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | [[Image:PCR_cycle.jpg]]
| |
| - | </center>
| |
| | <br> | | <br> |
| - | <br>
| |
| - | <br>
| |
| - |
| |
| - | === STAT1 (submission) ===
| |
| - |
| |
| - | <center>
| |
| - | [[Image:PCR_STAT1.jpg]]
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Primer</b></td><td><b>Direction</b></td><td><b>Sequences</b></td><td><b>Length</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>STAT_F</td><td>Forward</td><td>5’-ATGTCTCAGTGGTACGAACTTCAG-3’</td><td>24bp</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>STAT_R</td><td>Reverse</td><td>5’-TTACACTTCAGACACAGAAATCAAC-3’</td><td>25bp</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Materials</b></td><td><b>Amount</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>STAT1(KRIBB) (template)</td><td>150pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>STAT_F</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>STAT_R</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>dNTP (dNTP mixture 2.5mM TaKaRa)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)</td><td>0.25ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>10x buffer (10x cloned Pfu Reaction buffer)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>DW (3rd sterile water)</td><td>rest</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Total</td><td>50ul</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | [[Image:PCR_cycle.jpg]]
| |
| - | </center>
| |
| - | <br>
| |
| - | <br>
| |
| - | <br>
| |
| - |
| |
| - | === Fusion Antibody Receptor (Submission) ===
| |
| - |
| |
| - | <center>
| |
| - | [[Image:PCR_FGFR.jpg]]
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Primer</b></td><td><b>Direction</b></td><td><b>Sequences</b></td><td><b>Length</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>SIG_F</td><td>Forward</td><td>5’-ATGGTCTTTTTAAATTCCTCTCCC-3’</td><td>24bp</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Ab_R</td><td>Reverse</td><td>5’-GGGGCTGTTGTTTTGGCTGAGG-3’</td><td>22bp</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Materials</b></td><td><b>Amount</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Fusion antibody receptor T vector (template)</td><td>150pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>SIG_F</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Ab_R</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>dNTP (dNTP mixture 2.5mM TaKaRa)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)</td><td>0.25ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>10x buffer (10x cloned Pfu Reaction buffer)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>DW (3rd sterile water)</td><td>rest</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Total</td><td>50ul</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | [[Image:PCR_cycle.jpg]]
| |
| - | </center>
| |
| - | <br>
| |
| - | <br>
| |
| - | <br>
| |
| - |
| |
| - | === Signal Peptide (Submission) ===
| |
| - |
| |
| - | <center>
| |
| - | [[Image:PCR_FGFR.jpg]]
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Primer</b></td><td><b>Direction</b></td><td><b>Sequences</b></td><td><b>Length</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>SIG_F</td><td>Forward</td><td>5’-ATGGTCTTTTTAAATTCCTCTCCC-3’</td><td>24bp</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>SIG_R</td><td>Reverse</td><td>5’-AGCCGCCACCAACCGAGTAGAAA-3’</td><td>23bp</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Materials</b></td><td><b>Amount</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Fusion antibody receptor T vector (template)</td><td>150pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>SIG_F</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>SIG_R</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>dNTP (dNTP mixture 2.5mM TaKaRa)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)</td><td>0.25ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>10x buffer (10x cloned Pfu Reaction buffer)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>DW (3rd sterile water)</td><td>rest</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Total</td><td>50ul</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | [[Image:PCR_cycle.jpg]]
| |
| - | </center>
| |
| - | <br>
| |
| - | <br>
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| - | <br>
| |
| - |
| |
| - | === Ig-like (Submission) ===
| |
| - |
| |
| - | <center>
| |
| - | [[Image:PCR_FGFR.jpg]]
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| - |
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| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Primer</b></td><td><b>Direction</b></td><td><b>Sequences</b></td><td><b>Length</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>IG_F</td><td>Forward</td><td>5’-AGGCCGTCCCCGACCTTGCCTG-3’</td><td>22bp</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>IG_R</td><td>Reverse</td><td>5’-GGAGTCATCATCATCATCATCATC-3’</td><td>24bp</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
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| - | <br>
| |
| - |
| |
| - | <table border=1>
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| - | <tr align=center>
| |
| - | <td><b>Materials</b></td><td><b>Amount</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Fusion antibody receptor T vector (template)</td><td>150pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>IG_F</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>IG_R</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>dNTP (dNTP mixture 2.5mM TaKaRa)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)</td><td>0.25ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>10x buffer (10x cloned Pfu Reaction buffer)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>DW (3rd sterile water)</td><td>rest</td>
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| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Total</td><td>50ul</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | [[Image:PCR_cycle.jpg]]
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| - | </center>
| |
| - | <br>
| |
| - | <br>
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| - | <br>
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| - |
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| - | === Signal Peptide + Ig-like (Submission) ===
| |
| - |
| |
| - | <center>
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| - | [[Image:PCR_FGFR.jpg]]
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| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Primer</b></td><td><b>Direction</b></td><td><b>Sequences</b></td><td><b>Length</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>SIG_F</td><td>Forward</td><td>5’-ATGGTCTTTTTAAATTCCTCTCCC-3’</td><td>24bp</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>IG_R</td><td>Reverse</td><td>5’-GGAGTCATCATCATCATCATCATC-3’</td><td>24bp</td>
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| - | </tr>
| |
| - | </table>
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| - |
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| - | <br>
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| - |
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| - | <table border=1>
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| - | <tr align=center>
| |
| - | <td><b>Materials</b></td><td><b>Amount</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Fusion antibody receptor T vector (template)</td><td>150pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>SIG_F</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>IG_R</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>dNTP (dNTP mixture 2.5mM TaKaRa)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)</td><td>0.25ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>10x buffer (10x cloned Pfu Reaction buffer)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>DW (3rd sterile water)</td><td>rest</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Total</td><td>50ul</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | [[Image:PCR_cycle.jpg]]
| |
| - | </center>
| |
| - | <br>
| |
| - | <br>
| |
| - | <br>
| |
| - |
| |
| - | === Antibody (Submission) ===
| |
| - |
| |
| - | <center>
| |
| - | [[Image:PCR_FGFR.jpg]]
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Primer</b></td><td><b>Direction</b></td><td><b>Sequences</b></td><td><b>Length</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Ab_F</td><td>Forward</td><td>5’-ACCCAGTCTCCAGCAATCATGTC-3’</td><td>23bp</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Ab_R</td><td>Reverse</td><td>5’-GGGGCTGTTGTTTTGGCTGAGG-3’</td><td>22bp</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Materials</b></td><td><b>Amount</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Fusion antibody receptor T vector (template)</td><td>150pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Ab_F</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Ab_R</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>dNTP (dNTP mixture 2.5mM TaKaRa)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)</td><td>0.25ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>10x buffer (10x cloned Pfu Reaction buffer)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>DW (3rd sterile water)</td><td>rest</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Total</td><td>50ul</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | [[Image:PCR_cycle.jpg]]
| |
| - | </center>
| |
| - | <br>
| |
| - | <br>
| |
| - | <br>
| |
| - |
| |
| - | === V_Fusion Antibody Receptor (Expression) ===
| |
| - |
| |
| - | <center>
| |
| - | [[Image:PCR_FGFR.jpg]]
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Primer</b></td><td><b>Direction</b></td><td><b>Sequences</b></td><td><b>Length</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>SIG_F</td><td>Forward</td><td>5’-ATGGTCTTTTTAAATTCCTCTCCC-3’</td><td>24bp</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Linker_R</td><td>Reverse</td><td>5’-CTCTCTTCCAGGGCTTCCAGAACGGGGCTGTTGTTTTGGCTGAGGA-3’</td><td>46(23+23)bp</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Materials</b></td><td><b>Volume</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Fusion antibody receptor T vector (template)</td><td></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>SIG_F</td><td></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Linker_R</td><td></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>dNTP (dNTP mixture 2.5mM TaKaRa)</td><td></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)</td><td></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>10x buffer (10x cloned Pfu Reaction buffer)</td><td></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>DW (3rd sterile water)</td><td></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Total</td><td>50ul</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | [[Image:PCR_cycle.jpg]]
| |
| - | </center>
| |
| - | <br>
| |
| - | <br>
| |
| - | <br>
| |
| - |
| |
| - | === B_FGFR1 (Submission) ===
| |
| - |
| |
| - | <center>
| |
| - | [[Image:PCR_FGFR.jpg]]
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Primer</b></td><td><b>Direction</b></td><td><b>Sequences</b></td><td><b>Length</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>B_FGFR_F</td><td>Forward</td><td>5’-GTTCTGGAAGCCCTGGAAGAGAG-3’</td><td>23bp</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>B_FGFR_R</td><td>Reverse</td><td>5’-TCAGCGGCGTTTGAGTCCGCCAT-3’</td><td>23bp</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Materials</b></td><td><b>Volume</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>FGFR1(KRIBB) (template)</td><td></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>B_FGFR_F</td><td></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>B_FGFR_R</td><td></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>dNTP (dNTP mixture 2.5mM TaKaRa)</td><td></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)</td><td></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>10x buffer (10x cloned Pfu Reaction buffer)</td><td></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>DW (3rd sterile water)</td><td></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Total</td><td>50ul</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | [[Image:PCR_cycle.jpg]]
| |
| - | </center>
| |
| - | <br>
| |
| - | <br>
| |
| - | <br>
| |
| - |
| |
| - | === VB_FGFR1 (Expression) ===
| |
| - |
| |
| - | <center>
| |
| - | [[Image:PCR_FGFR.jpg]]
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Primer</b></td><td><b>Direction</b></td><td><b>Sequences</b></td><td><b>Length</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Linker_F</td><td>Forward</td><td>5’-TCCTCAGCCAAAACAACAGCCCCGTTCTGGAAGCCCTGGAAGAGAG-3’</td><td>46(23+23)bp</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>B_FGFR_R</td><td>Reverse</td><td>5’-TCAGCGGCGTTTGAGTCCGCCAT-3’</td><td>23bp</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Materials</b></td><td><b>Amount</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>FGFR1(KRIBB) (template)</td><td>150pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Linker_F</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>B_FGFR_R</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>dNTP (dNTP mixture 2.5mM TaKaRa)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)</td><td>0.25ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>10x buffer (10x cloned Pfu Reaction buffer)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>DW (3rd sterile water)</td><td>rest</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Total</td><td>50ul</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | [[Image:PCR_cycle.jpg]]
| |
| - | </center>
| |
| - | <br>
| |
| - | <br>
| |
| - | <br>
| |
| - |
| |
| - | === Fusion Antibody Receptor (Expression) ===
| |
| - |
| |
| - | <center>
| |
| - | [[Image:PCR_FGFR.jpg]]
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Primer</b></td><td><b>Direction</b></td><td><b>Sequences</b></td><td><b>Length</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>SIG_F</td><td>Forward</td><td>5’-ATGGTCTTTTTAAATTCCTCTCCC-3’</td><td>24bp</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>B_FGFR_R</td><td>Reverse</td><td>5’-TCAGCGGCGTTTGAGTCCGCCAT-3’</td><td>23bp</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Materials</b></td><td><b>Amount</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>V_Fusion antibody receptor (template)</td><td>150pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>VB_FGFR1(2+3) (template)</td><td>150pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>SIG_F</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>B_FGFR_R</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>dNTP (dNTP mixture 2.5mM TaKaRa)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)</td><td>0.25ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>10x buffer (10x cloned Pfu Reaction buffer)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>DW (3rd sterile water)</td><td>rest</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Total</td><td>50ul</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | [[Image:PCR_cycle.jpg]]
| |
| - | </center>
| |
| - | <br>
| |
| - | <br>
| |
| - | <br>
| |
| - |
| |
| - | === 5’UTR (expression) ===
| |
| - |
| |
| - | <center>
| |
| - | [[Image:PCR_HR.jpg]]
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Primer</b></td><td><b>Direction</b></td><td><b>Sequences</b></td><td><b>Length</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>HR_F</td><td>Forward</td><td>5’-AAACCAGTTTTGCCAAAACAAC-3’</td><td>22bp</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>UF_LinkR</td><td>Reverse</td><td>5’ –GAGAGGAATTTAAAAAGACCATAAACAACATTTTTCTTTTTTAC - 3’</td><td>44bp</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Materials</b></td><td><b>Amount</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>S.pombe genomic DNA (template)</td><td>150pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>HR_F</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>UF_LinkR</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>dNTP (dNTP mixture 2.5mM TaKaRa)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)</td><td>0.25ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>10x buffer (10x cloned Pfu Reaction buffer)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>DW (3rd sterile water)</td><td>rest</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Total</td><td>50ul</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | [[Image:PCR_cycle.jpg]]
| |
| - | </center>
| |
| - | <br>
| |
| - | <br>
| |
| - | <br>
| |
| - |
| |
| - | === 3’UTR (expression) ===
| |
| - |
| |
| - | <center>
| |
| - | [[Image:PCR_HR.jpg]]
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Primer</b></td><td><b>Direction</b></td><td><b>Sequences</b></td><td><b>Length</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>HU_LINKF (HA2 + 3’UTR)</td><td>Forward</td><td>5’-CCATATGACCCAGATTACGCTTGGAAATTAAATCGTGCGTAAG-3’</td><td>43bp</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>HR_R</td><td>Reverse</td><td>5’ – GCGTTCTTTTTTAATTCTGAATTAA – 3’</td><td>25bp</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Materials</b></td><td><b>Amount</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>S.pombe genomic DNA (template)</td><td>150pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>HU_LINKF (HA2 + 3’UTR)</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>HR_R</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>dNTP (dNTP mixture 2.5mM TaKaRa)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)</td><td>0.25ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>10x buffer (10x cloned Pfu Reaction buffer)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>DW (3rd sterile water)</td><td>rest</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Total</td><td>50ul</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | [[Image:PCR_cycle.jpg]]
| |
| - | </center>
| |
| - | <br>
| |
| - | <br>
| |
| - | <br>
| |
| - |
| |
| - | === Selection Marker (expression) ===
| |
| - |
| |
| - | <center>
| |
| - | [[Image:PCR_GAS.jpg]]
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Primer</b></td><td><b>Direction</b></td><td><b>Sequences</b></td><td><b>Length</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>HS_LINKF (HA1 + Selection Marker)</td><td>Forward</td><td>5’-CCATATGACCCAGATTACGCTAGCAAGCTTAAGCTTAGCTAC-3’</td><td>42bp</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>SH_LINKR</td><td>Reverse</td><td>5’-GTCAGGAACATCGTATGGGTAGACCTGCAGAAGCTTAAGCTTG-3’</td><td>43bp</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Materials</b></td><td><b>Amount</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>pREP42-GFP vector (template)</td><td>150pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>HS_LINKF (HA1 + Selection Marker)</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>SH_LINKR</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>dNTP (dNTP mixture 2.5mM TaKaRa)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)</td><td>0.25ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>10x buffer (10x cloned Pfu Reaction buffer)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>DW (3rd sterile water)</td><td>rest</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Total</td><td>50ul</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | [[Image:PCR_cycle.jpg]]
| |
| - | </center>
| |
| - | <br>
| |
| - | <br>
| |
| - | <br>
| |
| - |
| |
| - | === Core promoter + GFP (expression) ===
| |
| - |
| |
| - | <center>
| |
| - | [[Image:PCR_GAS.jpg]]
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Primer</b></td><td><b>Direction</b></td><td><b>Sequences</b></td><td><b>Length</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>PC_LINKF (Proximal + Core)</td><td>Forward</td><td>5’–CAGAATAGACACACGGGCCGACATTGAAGATATATAAAGGAAG–3’</td><td>43bp</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>CGFP_R (Core promoter + GFP)</td><td>Reverse</td><td>3’-<font color = blue>CGA</font><font color = red>CATATG</font><font color = blue>CTT</font>GTACAGCTCG-5’</td><td>22bp</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Materials</b></td><td><b>Amount</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>pREP42-GFP vector (template)</td><td>150pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>PC_LINKF (Proximal + Core)</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>CGFP_R (Core promoter + GFP)</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>dNTP (dNTP mixture 2.5mM TaKaRa)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)</td><td>0.25ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>10x buffer (10x cloned Pfu Reaction buffer)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>DW (3rd sterile water)</td><td>rest</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Total</td><td>50ul</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | [[Image:PCR_cycle.jpg]]
| |
| - | </center>
| |
| - | <br>
| |
| - | <br>
| |
| - | <br>
| |
| - |
| |
| - | === Proximal promoter (expression) ===
| |
| - |
| |
| - | <center>
| |
| - | [[Image:PCR_GAS.jpg]]
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Primer</b></td><td><b>Direction</b></td><td><b>Sequences</b></td><td><b>Length</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>PP_F (Proximal promoter)</td><td>Forward</td><td>5’-<font color = blue>CGC</font><font color = red>CTGCAG</font><font color = blue>GCG</font>AATAGCTGG–3’</td><td>41bp</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>PC_LINKR</td><td>Reverse</td><td>5’–CTTCCTTTATATATCTTCAATGTCGGCCCGTGTGTCTATTCTG–3’</td><td>43bp</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Materials</b></td><td><b>Amount</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Proximal promoter T vector (template)</td><td>150pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>PP_F (Proximal promoter)</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>PC_LINKR</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>dNTP (dNTP mixture 2.5mM TaKaRa)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)</td><td>0.25ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>10x buffer (10x cloned Pfu Reaction buffer)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>DW (3rd sterile water)</td><td>rest</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Total</td><td>50ul</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | [[Image:PCR_cycle.jpg]]
| |
| - | </center>
| |
| - | <br>
| |
| - | <br>
| |
| - | <br>
| |
| - |
| |
| - | === HA1 (expression) ===
| |
| - |
| |
| - | <center>
| |
| - | [[Image:PCR_HR.jpg]]
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Primer</b></td><td><b>Direction</b></td><td><b>Sequences</b></td><td><b>Length</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>FH_LINKF (FGFR + HA1)</td><td>Forward</td><td>5’-GCGGACTCAAACGCCGCTGATACCCATACGATGTTCCTGAC-3’</td><td>41bp</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>HS_LINKR</td><td>Reverse</td><td>5’-GTAGCTAAGCTTAAGCTTGCTAGCGTAATCTGGGTCATATGG-3’</td><td>42bp</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Materials</b></td><td><b>Amount</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>HA T vector (template)</td><td>150pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>FH_LINKF (FGFR + HA1)</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>HS_LINKR</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>dNTP (dNTP mixture 2.5mM TaKaRa)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)</td><td>0.25ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>10x buffer (10x cloned Pfu Reaction buffer)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>DW (3rd sterile water)</td><td>rest</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Total</td><td>50ul</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | [[Image:PCR_cycle.jpg]]
| |
| - | </center>
| |
| - | <br>
| |
| - | <br>
| |
| - | <br>
| |
| - |
| |
| - | === HA2 (expression) ===
| |
| - |
| |
| - | <center>
| |
| - | [[Image:PCR_HR.jpg]]
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Primer</b></td><td><b>Direction</b></td><td><b>Sequences</b></td><td><b>Length</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>SH_LINKF (Selection Marker + HA2)</td><td>Forward</td><td>5’-CAAGCTTAAGCTTCTGCAGGTCTACCCATACGATGTTCCTGAC-3’</td><td>43bp</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>HU_LINKR</td><td>Reverse</td><td>5’-CTTACGCACGATTTAATTTCCAAGCGTAATCTGGGTCATATGG-3’</td><td>43bp</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Materials</b></td><td><b>Amount</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>HA T vector (template)</td><td>150pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>SH_LINKF (Selection Marker + HA2)</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>HU_LINKR</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>dNTP (dNTP mixture 2.5mM TaKaRa)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)</td><td>0.25ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>10x buffer (10x cloned Pfu Reaction buffer)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>DW (3rd sterile water)</td><td>rest</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Total</td><td>50ul</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | [[Image:PCR_cycle.jpg]]
| |
| - | </center>
| |
| - | <br>
| |
| - | <br>
| |
| - | <br>
| |
| - |
| |
| - | === Fusion antibody receptor (expression) ===
| |
| - |
| |
| - | <center>
| |
| - | [[Image:PCR_HR.jpg]]
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Primer</b></td><td><b>Direction</b></td><td><b>Sequences</b></td><td><b>Length</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>UF_LinkF (5’UTR+FGFR Linker)</td><td>Forward</td><td>5’-GTAAAAAAGAAAAATGTTGTTTATGGTCTTTTTAAATTCCTCTC-3’</td><td>44bp</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>FH_LINKR</td><td>Reverse</td><td>5’-GTCAGGAACATCGTATGGGTATCAGCGGCGTTTGAGTCCGC-3’</td><td>41bp</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Materials</b></td><td><b>Amount</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Fusion Antibody receptor (template)</td><td>150pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>UF_LinkF (5’UTR+FGFR Linker)</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>FH_LINKR</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>dNTP (dNTP mixture 2.5mM TaKaRa)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)</td><td>0.25ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>10x buffer (10x cloned Pfu Reaction buffer)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>DW (3rd sterile water)</td><td>rest</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Total</td><td>50ul</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | [[Image:PCR_cycle.jpg]]
| |
| - | </center>
| |
| - | <br>
| |
| - | <br>
| |
| - | <br>
| |
| - |
| |
| - | === 5’UTR + Fusion Antibody receptor (expression) ===
| |
| - |
| |
| - | <center>
| |
| - | [[Image:PCR_HR.jpg]]
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Primer</b></td><td><b>Direction</b></td><td><b>Sequences</b></td><td><b>Length</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>HR_F (for homologous recombinant)</td><td>Forward</td><td>5’-AAACCAGTTTTGCCAAAACAAC-3’</td><td>22bp</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>FH_LINKR</td><td>Reverse</td><td>5’-GTCAGGAACATCGTATGGGTATCAGCGGCGTTTGAGTCCGC-3’</td><td>41bp</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Materials</b></td><td><b>Amount</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>5'UTR (template)</td><td>150pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Fusion Antibody receptor (template)</td><td>150pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>FH_LINKF (FGFR + HA1)</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>SH_LINKR</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>dNTP (dNTP mixture 2.5mM TaKaRa)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)</td><td>0.25ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>10x buffer (10x cloned Pfu Reaction buffer)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>DW (3rd sterile water)</td><td>rest</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Total</td><td>50ul</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | [[Image:PCR_cycle.jpg]]
| |
| - | </center>
| |
| - | <br>
| |
| - | <br>
| |
| - | <br>
| |
| - |
| |
| - | === HA1 + Selection Marker (expression) ===
| |
| - |
| |
| - | <center>
| |
| - | [[Image:PCR_HR.jpg]]
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Primer</b></td><td><b>Direction</b></td><td><b>Sequences</b></td><td><b>Length</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>FH_LINKF (FGFR + HA1)</td><td>Forward</td><td>5’-GCGGACTCAAACGCCGCTGATACCCATACGATGTTCCTGAC-3’</td><td>41bp</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>SH_LINKR</td><td>Reverse</td><td>5’-GTCAGGAACATCGTATGGGTAGACCTGCAGAAGCTTAAGCTTG-3’</td><td>43bp</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Materials</b></td><td><b>Amount</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>HA1 (template)</td><td>150pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Selection Marker (template)</td><td>150pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>FH_LINKF (FGFR + HA1)</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>SH_LINKR</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>dNTP (dNTP mixture 2.5mM TaKaRa)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)</td><td>0.25ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>10x buffer (10x cloned Pfu Reaction buffer)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>DW (3rd sterile water)</td><td>rest</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Total</td><td>50ul</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | [[Image:PCR_cycle.jpg]]
| |
| - | </center>
| |
| - | <br>
| |
| - | <br>
| |
| - | <br>
| |
| - |
| |
| - | === HA2 + 3'UTR (expression) ===
| |
| - |
| |
| - | <center>
| |
| - | [[Image:PCR_HR.jpg]]
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Primer</b></td><td><b>Direction</b></td><td><b>Sequences</b></td><td><b>Length</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>SH_LINKF (Selection Marker + HA2)</td><td>Forward</td><td>5’-CAAGCTTAAGCTTCTGCAGGTCTACCCATACGATGTTCCTGAC-3’</td><td>43bp</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>HR_R</td><td>Reverse</td><td>5’ – GCGTTCTTTTTTAATTCTGAATTAA – 3’</td><td>25bp</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Materials</b></td><td><b>Amount</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>HA2 (template)</td><td>150pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>3’UTR (template)</td><td>150pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>SH_LINKF (Selection Marker + HA2)</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>HR_R</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>dNTP (dNTP mixture 2.5mM TaKaRa)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)</td><td>0.25ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>10x buffer (10x cloned Pfu Reaction buffer)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>DW (3rd sterile water)</td><td>rest</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Total</td><td>50ul</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | [[Image:PCR_cycle.jpg]]
| |
| - | </center>
| |
| - | <br>
| |
| - | <br>
| |
| - | <br>
| |
| - |
| |
| - | === 5’UTR - Fusion Antibody receptor + HA1 - Selection Marker (expression) ===
| |
| - |
| |
| - | <center>
| |
| - | [[Image:PCR_HR.jpg]]
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Primer</b></td><td><b>Direction</b></td><td><b>Sequences</b></td><td><b>Length</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>HR_F(for homologous recombinant)</td><td>Forward</td><td>5’-AAACCAGTTTTGCCAAAACAAC-3’</td><td>22bp</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>SH_LINKR</td><td>Reverse</td><td>5’-GTCAGGAACATCGTATGGGTAGACCTGCAGAAGCTTAAGCTTG-3’</td><td>43bp</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Materials</b></td><td><b>Amount</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>5’UTR - Fusion Antibody receptor (template)
| |
| - | </td><td>150pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>HA1 - Selection Marker (template)
| |
| - | </td><td>150pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>HR_F (for homologous recombinant)</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>SH_LINKR</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>dNTP (dNTP mixture 2.5mM TaKaRa)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)</td><td>0.25ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>10x buffer (10x cloned Pfu Reaction buffer)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>DW (3rd sterile water)</td><td>rest</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Total</td><td>50ul</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | [[Image:PCR_cycle.jpg]]
| |
| - | </center>
| |
| - | <br>
| |
| - | <br>
| |
| - | <br>
| |
| - |
| |
| - | === Homologous Recombinant (expression) ===
| |
| - |
| |
| - | <center>
| |
| - | [[Image:PCR_HR.jpg]]
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Primer</b></td><td><b>Direction</b></td><td><b>Sequences</b></td><td><b>Length</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>HR_F(for homologous recombinant)</td><td>Forward</td><td>5’-AAACCAGTTTTGCCAAAACAAC-3’</td><td>22bp</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>HR_R</td><td>Reverse</td><td>5’ – GCGTTCTTTTTTAATTCTGAATTAA – 3’</td><td>25bp</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | <br>
| |
| - |
| |
| - | <table border=1>
| |
| - | <tr align=center>
| |
| - | <td><b>Materials</b></td><td><b>Amount</b></td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>HA2 + 3'UTR (template)
| |
| - | </td><td>150pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>5’UTR - Fusion Antibody receptor - HA1 - Selection Marker (template)
| |
| - | </td><td>150pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>HR_F (for homologous recombinant)</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>HR_R</td><td>50pmole</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>dNTP (dNTP mixture 2.5mM TaKaRa)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE)</td><td>0.25ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>10x buffer (10x cloned Pfu Reaction buffer)</td><td>5ul</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>DW (3rd sterile water)</td><td>rest</td>
| |
| - | </tr>
| |
| - | <tr align=center>
| |
| - | <td>Total</td><td>50ul</td>
| |
| - | </tr>
| |
| - | </table>
| |
| - |
| |
| - | [[Image:PCR_cycle.jpg]]
| |
| - | </center>
| |
| - | <br>
| |
| - | <br>
| |
| - | <br>
| |
| - |
| |
| - | == September next ==
| |
| - |
| |
| - |
| |
| - | </tr>
| |
| - |
| |
| - | </table>
| |
"
