Team:KAIST-Korea/Notebook/Diary/September

From 2010.igem.org

(Difference between revisions)
(→5’UTR - Fusion Antibody receptor + HA1 - Selection Marker (expression))
(→September 2 ~ September 28)
 
(73 intermediate revisions not shown)
Line 1: Line 1:
__NOTOC__
__NOTOC__
{{:Team:KAIST/header}}
{{:Team:KAIST/header}}
-
<table width="100%" border="0">
+
<table width="100%" border="0" cellpadding = "20px">
<tr>
<tr>
Line 9: Line 9:
<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>
Line 43: Line 43:
</tr>
</tr>
</table>
</table>
 +
== September 1 ==
== September 1 ==
pREP41 vector has arrived.
pREP41 vector has arrived.
Line 67: Line 68:
-
==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>Volume</b></td>
 
-
</tr>
 
-
<tr align=center>
 
-
<td>STAT1(KRIBB) (template)</td><td></td>
 
-
</tr>
 
-
<tr align=center>
 
-
<td>V_STAT_F</td><td></td>
 
-
</tr>
 
-
<tr align=center>
 
-
<td>V_STAT_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>
 
-
<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>Volume</b></td>
 
-
</tr>
 
-
<tr align=center>
 
-
<td>STAT1(KRIBB) (template)</td><td></td>
 
-
</tr>
 
-
<tr align=center>
 
-
<td>STAT_F</td><td></td>
 
-
</tr>
 
-
<tr align=center>
 
-
<td>STAT_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>
 
-
 
-
=== 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>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>Ab_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>
 
-
 
-
=== 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>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>SIG_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>
 
-
 
-
=== Ig-like (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>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>
 
-
 
-
<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>IG_F</td><td></td>
 
-
</tr>
 
-
<tr align=center>
 
-
<td>IG_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>
 
-
 
-
=== Signal Peptide + Ig-like (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>IG_R</td><td>Reverse</td><td>5’-GGAGTCATCATCATCATCATCATC-3’</td><td>24bp</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>IG_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>
 
-
 
-
=== 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>Volume</b></td>
 
-
</tr>
 
-
<tr align=center>
 
-
<td>Fusion antibody receptor T vector (template)</td><td></td>
 
-
</tr>
 
-
<tr align=center>
 
-
<td>Ab_F</td><td></td>
 
-
</tr>
 
-
<tr align=center>
 
-
<td>Ab_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>
 
-
 
-
=== V_Fusion Antibody Receptor (Expression) ===
 
-
=== B_FGFR1 (Submission) ===
 
-
=== VB_FGFR1 (Expression) ===
 
-
=== Fusion Antibody Receptor (Expression) ===
 
-
=== 5’UTR (expression) ===
 
-
=== 3’UTR (expression) ===
 
-
=== Selection Marker (expression) ===
 
-
=== Core promoter + GFP (expression) ===
 
-
=== Proximal promoter (expression) ===
 
-
=== HA1 (expression) ===
 
-
=== HA2 (expression) ===
 
-
=== Fusion antibody receptor (expression) ===
 
-
=== 5’UTR + Fusion Antibody receptor (expression) ===
 
-
=== 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>Volume</b></td>
 
-
</tr>
 
-
<tr align=center>
 
-
<td>HA1 (template)</td><td></td>
 
-
</tr>
 
-
<tr align=center>
 
-
<td>Selection Marker (template)</td><td></td>
 
-
</tr>
 
-
<tr align=center>
 
-
<td>FH_LINKF (FGFR + HA1)</td><td></td>
 
-
</tr>
 
-
<tr align=center>
 
-
<td>SH_LINKR</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>
 
-
 
-
=== 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>Volume</b></td>
 
-
</tr>
 
-
<tr align=center>
 
-
<td>HA2 (template)</td><td></td>
 
-
</tr>
 
-
<tr align=center>
 
-
<td>3’UTR (template)</td><td></td>
 
-
</tr>
 
-
<tr align=center>
 
-
<td>SH_LINKF (Selection Marker + HA2)</td><td></td>
 
-
</tr>
 
-
<tr align=center>
 
-
<td>HR_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>
 
-
 
-
=== 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>Volume</b></td>
 
-
</tr>
 
-
<tr align=center>
 
-
<td>5’UTR - Fusion Antibody receptor
 
-
</td><td></td>
 
-
</tr>
 
-
<tr align=center>
 
-
<td>HA1 - Selection Marker (template)
 
-
</td><td></td>
 
-
</tr>
 
-
<tr align=center>
 
-
<td>HR_F (for homologous recombinant)</td><td></td>
 
-
</tr>
 
-
<tr align=center>
 
-
<td>SH_LINKR</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>
 
-
 
-
=== 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>Volume</b></td>
 
-
</tr>
 
-
<tr align=center>
 
-
<td>HA2 + HR_R (template), 5’UTR - Fusion Antibody receptor + HA1 - Selection Marker (template)
 
-
</td><td></td>
 
-
</tr>
 
-
<tr align=center>
 
-
<td>HR_F (for homologous recombinant)</td><td></td>
 
-
</tr>
 
-
<tr align=center>
 
-
<td>HR_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>
 
-
 
-
 
-
 
-
[[Image:PCR_FGFR.jpg]]
 
-
[[Image:PCR_HR.jpg]]
 
-
[[Image:PCR_GAS.jpg]]
 
-
 
-
== September next ==
 
-
 
-
 
-
</tr>
 
-
 
-
</table>
 

Latest revision as of 07:21, 20 October 2010

 

September 1

pREP41 vector has arrived.

-> we will do E.coli transformation and increase its number.

pREP42-GFP vector has arrived (in E.coli)

-> will incubate a day and do spreading for further experiment.

Making culture media

  1. 500mL LB broth + agar 1.5% (7.5g)
  2. Do auto clave
  3. Cool it down until it reaches 50~60℃
  4. Add ampicillin(1000x 100mg/mL) and stir
  5. putting out bubbles on culture media
  6. pouling on petri dish and wait for 1 hour

Culture

  1. Competent cell + DN10A and leave it in ice for 30min
  2. Do heat shock for 45 sec and store it in ice for 2min
  3. Stabilize it in incubator for 30 to 40min
  4. Do spreading


September 2 ~ September 21

We designed PCR schedules and do PCR experiment. When PCR result was not satisfied, then we repeated PCR experiment. There are PCR details on PCR experiment menu.


Retrieved from "http://2010.igem.org/Team:KAIST-Korea/Notebook/Diary/September"