Team:KIT-Kyoto/c6h12o6

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<div id="doc-right" class="metro">
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<h2>Protocol</h2>
 +
 +
 +
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 13pt"><B>Miniprep</B></FONT></SPAN></FONT></P>
 +
 +
<P STYLE="margin-bottom: 0cm">
 +
</P>
 +
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Solution
 +
&#8544; (50 mM Tris-HCl and 10 mM EDTA, and 50 </FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">g/mL
 +
RNase A, pH 8.0 (25&#730;C))</FONT></SPAN></FONT></P>
 +
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Solution
 +
&#8545; (0.2 M NaOH and 1 % SDS)</FONT></SPAN></FONT></P>
 +
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Solution
 +
&#8546; (4 M guanidine hydrochloride and 0.5 M potassium acetate, pH 4.2)</FONT></SPAN></FONT></P>
 +
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Solution
 +
&#8547; (5 M guanidine hydrochloride, 20 mM Tris-HCl, and 38% ethanol, pH
 +
6.6 (25&#730;C))</FONT></SPAN></FONT></P>
 +
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Solution
 +
&#8548; (2 mM Tris-HCl and 80% ethanol, pH 7.5 (25&#730;C))</FONT></SPAN></FONT></P>
 +
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><FONT COLOR="#000000"><FONT SIZE=2 STYLE="font-size: 11pt"></FONT></FONT></FONT></P>
 +
 +
<br>
 +
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Pick
 +
up a single colony from plate and cultivate it overnight in 3ml LB
 +
medium containing appropriate antibiotic at 37&#730;C&#730;.</FONT></SPAN></FONT></P>
 +
 +
<P STYLE="margin-bottom: 0cm">&darr;</P>
 +
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Transfer
 +
the culture into a 1.5ml tube, centrifuge at 13,000 rpm for 1 minute,
 +
and discard the supernatant (2 times).</FONT></SPAN></FONT></P>
 +
 +
<P STYLE="margin-bottom: 0cm">&darr;</P>
 +
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
 +
250 </FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
 +
of Solution&#8544; to the pellet and mix well with vortex.</FONT></SPAN></FONT></P>
 +
 +
<P STYLE="margin-bottom: 0cm">&darr;</P>
 +
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
 +
250 </FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
 +
of Solution&#8545; and mix well by turning the tube upside down several
 +
times.</FONT></SPAN></FONT></P>
 +
 +
<P STYLE="margin-bottom: 0cm">&darr;</P>
 +
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
 +
350 </FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
 +
of Solution&#8546; and by turning the tube upside down several times.</FONT></SPAN></FONT></P>
 +
 +
<P STYLE="margin-bottom: 0cm">&darr;</P>
 +
 +
<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Centrifuge
 +
at 13,000 rpm for 5 minutes.</FONT></SPAN></FONT></P>
 +
 +
<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2">&darr;</P>
 +
 +
<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Transfer
 +
the supernatant (approx. 850</FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L)
 +
to a mini prep column.</FONT></SPAN></FONT></P>
 +
 +
<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2">&darr;</P>
 +
 +
<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Centrifuge
 +
at 13,000 rpm for 1 minute and discard the flow-through.</FONT></SPAN></FONT></P>
 +
 +
<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2">&darr;</P>
 +
 +
<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
 +
500 </FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
 +
of Solution&#8547;.</FONT></SPAN></FONT></P>
 +
 +
<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2">&darr;</P>
 +
 +
<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Centrifuge
 +
at 13,000 rpm for 1 minute and discard the flow-through.</FONT></SPAN></FONT></P>
 +
 +
<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2">&darr;</P>
 +
 +
<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
 +
700 </FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
 +
of Solution&#8548;.</FONT></SPAN></FONT></P>
 +
 +
<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2">&darr;</P>
 +
 +
<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Centrifuge
 +
at 13,000 rpm for 1 minute and discard the flow-through (2 times).</FONT></SPAN></FONT></P>
 +
 +
<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2">&darr;</P>
 +
 +
<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Set
 +
the column on a new 1.5ml tube and add 100 </FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
 +
of nuclease-free water.</FONT></SPAN></FONT></P>
 +
 +
<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2">&darr;</P>
 +
 +
<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Centrifuge
 +
at 13,000 rpm for 1 minute and collect plasmid DNA.</FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"> </SPAN></FONT></P>
 +
 +
 +
<BR>
 +
 +
<P STYLE="margin-bottom: 0cm; page-break-before: always"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 13pt"><B>Rapid
 +
check of the insert by colony cracking</B></FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm">
 +
</P>
 +
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
 +
45 </FONT>&micro;L<FONT SIZE=2 STYLE="font-size: 11pt"> of cracking
 +
solution(3% w/v SDS ,50mM Tris -base ,pH12.6) into each tube.  </FONT></SPAN></FONT></p>
 +
 +
<P STYLE="margin-bottom: 0cm">&darr;</P>
 +
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Suspend
 +
a small quantity of the colony in a tube using a sterilized
 +
toothpick.</FONT></SPAN></FONT></P>
 +
 +
<P STYLE="margin-bottom: 0cm">&darr;</P>
 +
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Incubate
 +
the tube at 65&#176;C for 10 minutes.</FONT></SPAN></FONT></P>
 +
 +
<P STYLE="margin-bottom: 0cm">&darr;</P>
 +
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
 +
a drop of 10x Loading Buffer.</FONT></SPAN></FONT></P>
 +
 +
<P STYLE="margin-bottom: 0cm">&darr;</P>
 +
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
 +
equal volume of phenol/chloroform.</FONT></SPAN></FONT></P>
 +
 +
<P STYLE="margin-bottom: 0cm">&darr;</P>
 +
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Mix
 +
with vortex.</FONT></SPAN></FONT></P>
 +
 +
<P STYLE="margin-bottom: 0cm">&darr;</P>
 +
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Centrifuge
 +
at 13,000rpm for 3 minutes.</FONT></SPAN></FONT></P>
 +
 +
<P STYLE="margin-bottom: 0cm">&darr;</P>
 +
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Apply
 +
the supernatant to 1% agarose gel electrophoresis.</FONT></SPAN></FONT></P>
 +
 +
<P STYLE="margin-bottom: 0cm">&darr;</P>
 +
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Stain
 +
the gel in ethidium bromide solution for 10 minutes.</FONT></SPAN></FONT></P>
 +
 +
<P STYLE="margin-bottom: 0cm">&darr;</P>
 +
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Plasmid
 +
DNA can be detected by UV-illuminator as a band between genomic DNA
 +
band and low molecular size RNAs.</FONT></SPAN></FONT></P>
 +
 +
<P STYLE="margin-bottom: 0cm"><BR>
 +
</P>
 +
 +
<UL>
 +
<LI><P STYLE="margin-bottom: 0cm; page-break-before: always"><FONT FACE=""><B>
 +
</B></FONT><FONT FACE=""><SPAN LANG="en-US"><B>DNA purification and
 +
precipitation</B></SPAN></FONT></P>
 +
</UL>
 +
 +
<P STYLE="margin-left: 0.74cm; margin-bottom: 0cm"><BR>
 +
</P>
 +
<OL>
 +
<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Mix
 +
350 &#181;L of sterilized water and 50 &#181;L of 3 M sodium
 +
acetate with DNA sample.</SPAN></FONT></P>
 +
<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Mix
 +
DNA solution with equal volume of phenol/chloroform by vortex.</SPAN></FONT></P>
 +
<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Centrifuge
 +
at 13,000 rpm for 5 min, then transfer the supernatant into a new
 +
tube.</SPAN></FONT></P>
 +
<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Add
 +
equal volume of 2-propanol, and mix by turning the tube upside down.</SPAN></FONT></P>
 +
<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Centrifuge
 +
at 14,500 rpm for 10 min at 4&#730;C.</SPAN></FONT></P>
 +
<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Carefully
 +
decant the supernatant.</SPAN></FONT></P>
 +
<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Add
 +
adequate volume of 70 % ethanol.</SPAN></FONT></P>
 +
<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Centrifuge
 +
at 14,500 rpm for 5 min at 4&#730;C.</SPAN></FONT></P>
 +
<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Carefully
 +
decant the supernatant.</SPAN></FONT></P>
 +
<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Dry
 +
in the desiccator under vacuum for 5 min.</SPAN></FONT></P>
 +
<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">You
 +
can get dried DNA pellet.</SPAN></FONT></P>
 +
</OL>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><FONT COLOR="#000000"></FONT></FONT></P>
 +
<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><BR>
 +
</P>
 +
<P STYLE="margin-bottom: 0cm; page-break-before: always"><BR>
 +
</P>
 +
<UL>
 +
<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><B>PCR</B></SPAN></FONT></P>
 +
</UL>
 +
<P STYLE="margin-left: 0.74cm; margin-bottom: 0cm"><BR>
 +
</P>
 +
<OL>
 +
<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Adjust
 +
the concentration of each primer to 100 pmol/&micro;L with
 +
sterilized water.</SPAN></FONT></P>
 +
<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Mix
 +
10&micro;L of forward and reverse primer solutions with 80 &micro;L
 +
H<SUB>2</SUB>O in a new tube (final primer concentration is 10
 +
pmol/&micro;L).</SPAN></FONT></P>
 +
<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Use
 +
1 &micro;L of primer mix for PCR.</SPAN></FONT></P>
 +
</OL>
 +
<P STYLE="margin-left: 1.48cm; margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Recipe
 +
for PRC is as follows:</SPAN></FONT></P>
 +
<TABLE WIDTH=206 BORDER=1 BORDERCOLOR="#00000a" CELLPADDING=7 CELLSPACING=0>
 +
 +
<TR VALIGN=TOP>
 +
<TD WIDTH=106>
 +
<P><FONT FACE=""><SPAN LANG="en-US">Buffer</SPAN></FONT></P>
 +
</TD>
 +
<TD WIDTH=70>
 +
<P><FONT FACE=""><SPAN LANG="en-US">50 &micro;L</SPAN></FONT></P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=TOP>
 +
<TD WIDTH=106>
 +
<P><FONT FACE=""><SPAN LANG="en-US">dNTP</SPAN></FONT></P>
 +
</TD>
 +
<TD WIDTH=70>
 +
<P><FONT FACE=""><SPAN LANG="en-US">20 &micro;L</SPAN></FONT></P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=TOP>
 +
<TD WIDTH=106>
 +
<P><FONT FACE=""><SPAN LANG="en-US">Primer mix</SPAN></FONT></P>
 +
</TD>
 +
<TD WIDTH=70>
 +
<P> <FONT FACE=""><SPAN LANG="en-US">1 &micro;L</SPAN></FONT></P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=TOP>
 +
<TD WIDTH=106>
 +
<P><FONT FACE=""><SPAN LANG="en-US">DNA sample</SPAN></FONT></P>
 +
</TD>
 +
<TD WIDTH=70>
 +
<P><FONT FACE=""><SPAN LANG="en-US">0.5 &micro;L</SPAN></FONT></P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=TOP>
 +
<TD WIDTH=106>
 +
<P><FONT FACE=""><SPAN LANG="en-US">KOD-FX</SPAN></FONT></P>
 +
</TD>
 +
<TD WIDTH=70>
 +
<P> <FONT FACE=""><SPAN LANG="en-US">2 &micro;L</SPAN></FONT></P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=TOP>
 +
<TD WIDTH=106>
 +
<P><FONT FACE=""><SPAN LANG="en-US">H<SUB>2</SUB>O</SPAN></FONT></P>
 +
</TD>
 +
<TD WIDTH=70>
 +
<P><FONT FACE=""><SPAN LANG="en-US">26.5 &micro;L</SPAN></FONT></P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=TOP>
 +
<TD WIDTH=106>
 +
<P><FONT FACE=""><SPAN LANG="en-US">total</SPAN></FONT></P>
 +
</TD>
 +
<TD WIDTH=70>
 +
<P><FONT FACE=""><SPAN LANG="en-US">100 &micro;L</SPAN></FONT></P>
 +
</TD>
 +
</TR>
 +
</TABLE>
 +
<P STYLE="margin-bottom: 0cm"><BR>
 +
</P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">KOD-FX</SPAN></FONT><FONT FACE=""></FONT><FONT FACE=""><SPAN LANG="en-US">(</SPAN></FONT><FONT FACE=""><FONT COLOR="#000000">Toyobo</FONT></FONT><FONT FACE=""><SPAN LANG="en-US">)</SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><BR>
 +
</P>
 +
<P ALIGN=LEFT STYLE="margin-bottom: 0cm; widows: 2; orphans: 2"><BR>
 +
</P>
 +
<P STYLE="margin-bottom: 0cm; page-break-before: always"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">SDS
 +
polyacrylamide gel electrophoresis (SDS-PAGE)</FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><BR>
 +
</P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt"><B>12.5%
 +
separation gel (recipe for a sheet of gel)</B></FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Mili
 +
Q water 2.59 ml </FONT></SPAN></FONT>
 +
</P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">acrylamide
 +
solution(</FONT><FONT COLOR="#000000"><FONT SIZE=2 STYLE="font-size: 11pt">30
 +
</FONT></FONT><FONT SIZE=2 STYLE="font-size: 11pt">%) 3.33 ml</FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">0.5M
 +
Tris (pH8.8) 2 ml </FONT></SPAN></FONT>
 +
</P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">10%SDS 80
 +
</FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
 +
</FONT></SPAN></FONT>
 +
</P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">10%APS 27
 +
</FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
 +
</FONT></SPAN></FONT>
 +
</P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">TEMED 4
 +
</FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
 +
</FONT></SPAN></FONT>
 +
</P>
 +
<P STYLE="margin-bottom: 0cm">&darr;</P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Apply
 +
the acrylamide solution mix to the PAGE glass plate.</FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm">&darr;</P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Deposit
 +
H</FONT><SUB><FONT SIZE=2 STYLE="font-size: 11pt">2</FONT></SUB><FONT SIZE=2 STYLE="font-size: 11pt">O
 +
carefully on the top of the acrylamide solution mix.</FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm">&darr;</P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Wait
 +
for 10 min for polymerization.</FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><BR>
 +
</P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt"><B>Stacking
 +
gel  </B></FONT></SPAN></FONT>
 +
</P>
 +
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Mili
 +
Q water 2.89 ml</FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Acrylamide
 +
0.79 ml</FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">0.5M
 +
Tris (pH 6.8) 1.25 ml</FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">10%
 +
SDS 50 </FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L</FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">10%APS
 +
17 </FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L</FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">TEMED
 +
5 </FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L</FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm">&darr;</P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">After
 +
the polymerization of the separation gel, remove the H</FONT><SUB><FONT SIZE=2 STYLE="font-size: 11pt">2</FONT></SUB><FONT SIZE=2 STYLE="font-size: 11pt">O
 +
of the top layer.</FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm">&darr;</P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Apply
 +
stacking gel mix on the running gel and put the comb to make wells.</FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm">&darr;</P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">After
 +
the stacking gel has fully polymerized, remove the comb and rinse the
 +
top of the gel with H</FONT><SUB><FONT SIZE=2 STYLE="font-size: 11pt">2</FONT></SUB><FONT SIZE=2 STYLE="font-size: 11pt">O
 +
and then remove H</FONT><SUB><FONT SIZE=2 STYLE="font-size: 11pt">2</FONT></SUB><FONT SIZE=2 STYLE="font-size: 11pt">O.</FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><BR>
 +
</P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt"><B>Sample
 +
preparation</B></FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Collect
 +
bacterial cells.</FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm">&darr;</P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
 +
450ul of H</FONT><SUB><FONT SIZE=2 STYLE="font-size: 11pt">2</FONT></SUB><FONT SIZE=2 STYLE="font-size: 11pt">O
 +
and 50 </FONT><FONT SIZE=2 STYLE="font-size: 11pt">&#181;</FONT><FONT SIZE=2 STYLE="font-size: 11pt">L
 +
of FastBreak Cell Lysis Reagent (</FONT><FONT SIZE=2 STYLE="font-size: 11pt">Madison,
 +
Wisconsin, USA</FONT><FONT SIZE=2 STYLE="font-size: 11pt"> Promega).</FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm">&darr;</P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Mix
 +
them for 15minutes with shaker.</FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm">&darr;</P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Add
 +
100ul of 6</FONT></SPAN></FONT><FONT FACE=""><FONT SIZE=2 STYLE="font-size: 11pt">x</FONT></FONT><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">SDS-Sample
 +
buffer and mix with vortex.</FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm">&darr;</P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Heat
 +
samples in a heating block at 99&#176;C for 5 minutes.</FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><BR>
 +
</P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt"><B>Running
 +
samples</B></FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Set
 +
the gel on the electrophoresis apparatus and apply SDS running buffer
 +
(</FONT></SPAN></FONT><FONT FACE=""><FONT COLOR="#000000"><FONT SIZE=2 STYLE="font-size: 11pt">Tris 25mM,Glysine 191mM,SDS0.1%</FONT></FONT></FONT><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">)
 +
</FONT></SPAN></FONT>
 +
</P>
 +
<P STYLE="margin-bottom: 0cm">&darr;</P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Apply
 +
the samples and markers.</FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm">&darr;</P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Operate
 +
at constant current (25mA) for 65 minutes per sheet of gel.</FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><BR>
 +
</P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt"><B>Staining</B></FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Place
 +
the gel in stacking solution( </FONT></SPAN></FONT><FONT FACE=""><FONT COLOR="#000000"><FONT SIZE=2 STYLE="font-size: 11pt">50%ethanol,10%acetic acid</FONT></FONT></FONT><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">)
 +
and shake gently it for 5 minutes.</FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm">&darr;</P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Remove
 +
the stacking solution and add 100ml of staining solution (</FONT></SPAN></FONT><FONT FACE=""><FONT COLOR="#000000"><FONT SIZE=2 STYLE="font-size: 11pt">0.25% CBB R250、5%methanol、7.5%acetic acid</FONT></FONT></FONT><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">).</FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Warm
 +
the gel for 1 minute with a microwave (500W).</FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm">&darr;</P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><FONT SIZE=2 STYLE="font-size: 11pt">Remove
 +
the staining solution and rinse the gel with water.</FONT></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><BR>
 +
</P>
 +
<UL>
 +
<LI><P STYLE="margin-bottom: 0cm; page-break-before: always"><FONT FACE=""><B></B></FONT></P>
 +
<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><B>Isolation
 +
and purification of DNA bands</B></SPAN></FONT></P>
 +
</UL>
 +
<P STYLE="margin-left: 0.74cm; margin-bottom: 0cm"><BR>
 +
</P>
 +
<OL>
 +
<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Clip
 +
DNA band from agarose gel and transfer it into a 1.5 ml tube.</SPAN></FONT></P>
 +
<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Add
 +
H<SUB>2</SUB>O. Melt the gel at 65&#730;C in a heating block.</SPAN></FONT></P>
 +
<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Add
 +
equal volume of phenol. Mix well with vortex.</SPAN></FONT></P>
 +
<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Centrifuge
 +
at 13,000 rpm for 5 min.</SPAN></FONT></P>
 +
<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Transfer
 +
the supernatant into a new tube and mix with equal volume of
 +
phenol/chloroform.</SPAN></FONT></P>
 +
<LI><P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US">Perform
 +
the same method of DNA precipitation using 2-propanol.</SPAN></FONT></P>
 +
 +
 +
Blue gel<br>
 +
・50×TAE 100ml<br>
 +
・Agarose(nacalai tesque) 1.0g<br>
 +
・Gel indicator(Biodynamics laboratory) 200ul<br>
 +
 +
</OL>
 +
<P STYLE="margin-bottom: 0cm"><BR>
 +
</P>
 +
<P STYLE="margin-bottom: 0cm"><BR>
 +
</P>
 +
<P STYLE="margin-bottom: 0cm"><BR>
 +
</P>
 +
 +
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><B>&lt;Ligation&gt;</B></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><BR>
 +
</P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Dissolve
 +
the purified and dried insert DNA in 5&#181;L of H<SUB>2</SUB>O.</SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Dissolve
 +
the purified and dried vector DNA in 5&#181;L of H2O.</SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Mix
 +
the two solutions.</SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Add
 +
10&micro;L of Ligation Mix (</SPAN></FONT><FONT FACE=""><FONT COLOR="#000000">Wako</FONT></FONT><FONT FACE=""><SPAN LANG="en-US">)
 +
to it.</SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Incubate
 +
for 15 minutes at room temperature.</SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><BR>
 +
</P>
 +
<P STYLE="margin-bottom: 0cm"><BR>
 +
</P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE=""><SPAN LANG="en-US"><B>&lt;Transformation&gt;</B></SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><BR>
 +
</P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Add
 +
the ligation solution to competent cells (in a clean bench).</SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Place
 +
tubes on ice for 20 minutes.</SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Heat
 +
shock treatment at 42&#176;C for 35 seconds.</SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Place
 +
tubes on ice for 2 minutes.</SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Add
 +
1mL of LB medium into the tube (in a clean bench).</SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Incubate
 +
the samples for 20 minutes at 37&#176;C.</SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Centrifuge
 +
at 7,000rpm for 3 minutes.</SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Discard
 +
the most of supernatant, and spread the remaining cells and LD medium
 +
in the tube on the plates (in a clean bench).</SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><FONT FACE="">・</FONT><FONT FACE=""><SPAN LANG="en-US">Incubate
 +
plates overnight at 37&#176;C.</SPAN></FONT></P>
 +
<P STYLE="margin-bottom: 0cm"><BR>
 +
</P>
 +
 +
</div>
 +
 +
</div>
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
{{KIT-Kyoto}}
{{KIT-Kyoto}}
<html>
<html>
Line 6: Line 567:
<h2>August 1st</h2>
<h2>August 1st</h2>
-
 
+
<br>
-
<Table Border Cellspacing="0">
+
-
<Tr><Td></Td><Td>sample1</Td></Tr>
+
-
<Tr><Td>10ng/μL TNFAIP3</Td><Td>6μL</Td></Tr>
+
-
<Tr><Td>10× KOD plus buffer</Td><Td>10μL</Td></Tr>
+
-
<Tr><Td>2mM dNTPs</Td><Td>10μL</Td></Tr>
+
-
<Tr><Td>25mM MgSO<sub>4</sub></Td><Td>3.2μL</Td></Tr>
+
-
<Tr><Td>10P 5'primer</Td><Td>3μL</Td></Tr>
+
-
<Tr><Td>10P 3'primer</Td><Td>3μL</Td></Tr>
+
-
<Tr><Td>KOD plus</Td><Td>2μL</Td></Tr>
+
-
<Tr><Td>dH<sub>2</sub>O</Td><Td>62.8μL</Td></Tr>
+
-
<Tr><Td>total</Td><Td>100μL</Td></Tr>
+
-
</Table>
+
<br>
<br>
-
<Table Border Cellspacing="0">
+
Since it takes at least one month to establish transgenic flies, we decided to first construct the P-element plasmid for microinjection into Drosophia embryos.<br>
-
<Tr><Td>temperature</Td><Td>time</Td><Td>cycle</Td></Tr>
+
<br>
-
<Tr><Td>95°C</Td><Td>2min</Td><Td></Td></Tr>
+
① We performed PCR in the following conditions.<br>
-
<Tr><Td>95°C</Td><Td>15sec</Td><Td>25cycle</Td></Tr>
+
-
<Tr><Td>60°C</Td><Td>30sec</Td><Td>25cycle</Td></Tr>
+
-
<Tr><Td>68°C</Td><Td>2min30sec</Td><Td>25cycle</Td></Tr>
+
-
<Tr><Td>68°C</Td><Td>2min30sec</Td><Td></Td></Tr>
+
-
<Tr><Td>14°C</Td><Td>∞</Td><Td></Td></Tr>
+
-
</Table>
+
<br>
<br>
 +
(ⅰ)TNFAIP3<br>
 +
Composition for reaction
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
-
<Tr><Td></Td><Td>sample1</Td><Td>sample2</Td><Td>sample3</Td><Td>sample4</Td></Tr>
+
<Tr><Td>  </Td><Td> sample1 </Td></Tr>
-
<Tr><Td>10ng/μL API2-MALT1</Td><Td>2μL</Td><Td>2μL</Td><Td>1μL</Td><Td>1μL</Td></Tr>
+
<Tr><Td> 10ng/μL TNFAIP3 </Td><Td> 6μL </Td></Tr>
-
<Tr><Td>10× KOD plus buffer</Td><Td>2μL</Td><Td>2μL</Td><Td>2μL</Td><Td>2μL</Td></Tr>
+
<Tr><Td> 10× KOD plus buffer </Td><Td> 10μL </Td></Tr>
-
<Tr><Td>2mM dNTPs</Td><Td>2μL</Td><Td>2μL</Td><Td>2μL</Td><Td>2μL</Td></Tr>
+
<Tr><Td> 2mM dNTPs </Td><Td> 10μL </Td></Tr>
-
<Tr><Td>25mM MgSO<sub>4</sub></Td><Td>0.8μL</Td><Td>0.8μL</Td><Td>0.8μL</Td><Td>0.8μL</Td></Tr>
+
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 3.2μL </Td></Tr>
-
<Tr><Td>10P 5'primer</Td><Td>0.6μL</Td><Td>0.6μL</Td><Td>0.6μL</Td><Td>0.6μL</Td></Tr>
+
<Tr><Td> 10P 5'primer </Td><Td> 3μL </Td></Tr>
-
<Tr><Td>10P 3'primer</Td><Td>0.6μL</Td><Td>0.6μL</Td><Td>0.6μL</Td><Td>0.6μL</Td></Tr>
+
<Tr><Td> 10P 3'primer </Td><Td> 3μL </Td></Tr>
-
<Tr><Td>KOD plus</Td><Td>0.4μL</Td><Td>0.4μL</Td><Td>0.4μL</Td><Td>0.4μL</Td></Tr>
+
<Tr><Td> KOD plus </Td><Td> 2μL </Td></Tr>
-
<Tr><Td>dH<sub>2</sub>O</Td><Td>11.6μL</Td><Td>11.6μL</Td><Td>12.6μL</Td><Td>12.6μL</Td></Tr>
+
<Tr><Td> dH<sub>2</sub>O </Td><Td> 62.8μL </Td></Tr>
-
<Tr><Td>total</Td><Td>20μL</Td><Td>20μL</Td><Td>20μL</Td><Td>20μL</Td></Tr>
+
<Tr><Td> Total </Td><Td> 100μL </Td></Tr>
</Table>
</Table>
<br>
<br>
 +
Reaction conditions
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
-
<Tr><Td>temperature</Td><Td>time</Td><Td>cycle</Td></Tr>
+
<Tr><Td> temperature </Td><Td> time </Td><Td> cycle </Td></Tr>
-
<Tr><Td>95°C</Td><Td>2min</Td><Td></Td></Tr>
+
<Tr><Td> 95°C </Td><Td> 2min. </Td><Td>  </Td></Tr>
-
<Tr><Td>95°C</Td><Td>15sec</Td><Td>25cycle</Td></Tr>
+
<Tr><Td> 95°C </Td><Td> 15sec </Td><Td> 25cycle </Td></Tr>
-
<Tr><Td>50°C(sample1,3)or55°C(sample2,4)</Td><Td>30sec</Td><Td>25cycle</Td></Tr>
+
<Tr><Td> 60°C </Td><Td> 30sec </Td><Td> 25cycle </Td></Tr>
-
<Tr><Td>68°C</Td><Td>3min30sec</Td><Td>25cycle</Td></Tr>
+
<Tr><Td> 68°C </Td><Td> 2min30sec </Td><Td> 25cycle </Td></Tr>
-
<Tr><Td>68°C</Td><Td>3min30sec</Td><Td></Td></Tr>
+
<Tr><Td> 68°C </Td><Td> 2min30sec </Td><Td>  </Td></Tr>
-
<Tr><Td>14°C</Td><Td></Td><Td></Td></Tr>
+
<Tr><Td> 14°C </Td><Td> ∞ </Td><Td>  </Td></Tr>
</Table>
</Table>
 +
<br>
<br>
<br>
 +
<h2>August 2nd</h2>
<h2>August 2nd</h2>
 +
<br>
 +
<br>
 +
① PCR product check<br>
 +
<br>
 +
We run 0.7% agarose gel electorophoresis in the following conditions.
 +
Composition
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
-
<Tr><Td></Td><Td>1-2</Td><Td>1-3</Td></Tr>
+
<Tr><Td>  </Td><Td> 8/1 the attB TNFAIP3 PCR products </Td></Tr>
-
<Tr><Td>DNA sample</Td><Td>10μL</Td><Td>20μL</Td></Tr>
+
<Tr><Td> DNA sample </Td><Td> 10μL </Td></Tr>
-
<Tr><Td>6×Dye</Td><Td>2μL</Td><Td>4μL</Td></Tr>
+
<Tr><Td> 6×Dye </Td><Td> 2μL </Td></Tr>
-
<Tr><Td>total</Td><Td>12μL</Td><Td>25μL</Td></Tr>
+
<Tr><Td> total </Td><Td> 12μL </Td></Tr>
-
</Table>
+
</Table><br>
-
<br><br>
+
<br>
 +
 
 +
The order of sample application<br>
 +
Left : 1kb marker(5uL)<br>
 +
Right: attB TNFAIP3<br>
 +
<br>
 +
 
 +
<strong>Results</strong>
 +
 
 +
<br>
<img src="http://2012.igem.org/wiki/images/e/e0/0802.png" width="500" height="300">
<img src="http://2012.igem.org/wiki/images/e/e0/0802.png" width="500" height="300">
<br>
<br>
<br>
<br>
 +
 +
② DNA purification from gel<br>
 +
We electrophoresed in 0.7% agarose gel in the following conditions.<br>
 +
<br>
 +
 +
Composition
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
-
<Tr><Td></Td><Td>1-2</Td></Tr>
+
<Tr><Td>  </Td><Td> 8/1 attB TNFAIP3 PCRproduct </Td></Tr>
-
<Tr><Td>DNA sample</Td><Td>90μL</Td></Tr>
+
<Tr><Td> DNA sample </Td><Td> 90μL </Td></Tr>
-
<Tr><Td>6×Dye</Td><Td>18μL</Td></Tr>
+
<Tr><Td> 6×Dye </Td><Td> 18μL </Td></Tr>
-
<Tr><Td>total</Td><Td>108μL</Td></Tr>
+
<Tr><Td> total </Td><Td> 108μL </Td></Tr>
</Table>
</Table>
<br><br>
<br><br>
 +
We applied half of 108 uL of sample to each of two lanes of Agaroe gel with 5uL of 1kb marker.Then we detected the DNA band in the gel.<br><br>
 +
 +
<strong>Results</strong><br>
 +
<img src="http://2012.igem.org/wiki/images/1/19/0802-2.png" width="500" height="300">
<img src="http://2012.igem.org/wiki/images/1/19/0802-2.png" width="500" height="300">
 +
<br>
 +
 +
We purifed DNA from the gel by QIA Quick Gel Extraction Kit.<br>
<br><br>
<br><br>
-
<img src="http://2012.igem.org/wiki/images/0/0e/0802-3.png" width="500" height="300">
+
・Eatimation of the amount of attB TNFAIP3 DNA fragments by electrophoresis.<br>
-
<br><br>
+
Order of samples applied to the agarose gel<br>
-
<Table Border Cellspacing="0">
+
The samples were electrophoresed in 0.7% agarose gel in following order.<br>
-
<Tr><Td>10ng/μL API2-MALT1</Td><Td>5μL</Td></Tr>
+
Right to left:3uL of 1kb marker 1uL of 5 fold dilution of attB TNFAIP3 DNA fragments,1uL of 10 fold dilution of attB TNFAIP3 DNA fragments, 1uL of 20 fold dilution of attB TNFAIP3 DNA fragments, 1uL of 1kb marker.<br>
-
<Tr><Td>10×KOD plus buffer</Td><Td>10μL</Td></Tr>
+
-
<Tr><Td>2mM dNTPs</Td><Td>10μL</Td></Tr>
+
-
<Tr><Td>25mM MgSO<sub>4</sub></Td><Td>4μL</Td></Tr>
+
-
<Tr><Td>10P 5'primer</Td><Td>3μL</Td></Tr>
+
-
<Tr><Td>10P 3'primer</Td><Td>3μL</Td></Tr>
+
-
<Tr><Td>KOD plus</Td><Td>2μL</Td></Tr>
+
-
<Tr><Td>dH<sub>2</sub>O</Td><Td>63μL</Td></Tr>
+
-
<Tr><Td>total</Td><Td>100μL</Td></Tr>
+
-
</Table>
+
<br>
<br>
-
<Table Border Cellspacing="0">
+
<strong>Results</strong>
-
<Tr><Td>temperature</Td><Td>time</Td><Td>cycle</Td></Tr>
+
<br>
-
<Tr><Td>95°C</Td><Td>2min</Td><Td></Td></Tr>
+
<img src="http://2012.igem.org/wiki/images/0/0e/0802-3.png" width="500" height="300">
-
<Tr><Td>95°C</Td><Td>15sec</Td><Td>35cycle</Td></Tr>
+
<br>
-
<Tr><Td>55°C</Td><Td>30sec</Td><Td>35cycle</Td></Tr>
+
We have estimated amount of the attB TNFAIP3 DNA to be 80ng/uL.<br>
-
<Tr><Td>68°C</Td><Td>3min30sec</Td><Td>35cycle</Td></Tr>
+
<br>
-
<Tr><Td>68°C</Td><Td>3min30sec</Td><Td></Td></Tr>
+
-
<Tr><Td>14°C</Td><Td>∞</Td><Td></Td></Tr>
+
-
</Table>
+
<br>
<br>
-
<strong>Result</strong>
 
-
<br><br>
 
-
<img src="http://2012.igem.org/wiki/images/1/18/0802-4.png" width="500" height="300">
 
-
<br><br>
 
<h2>August 3rd</h2>
<h2>August 3rd</h2>
-
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td>attB TNFAIP3(80ng/μL)</Td><Td>1μL</Td></Tr>
 
-
<Tr><Td>pDONR(455ng/μL)</Td><Td>0.35μL</Td></Tr>
 
-
<Tr><Td>TE buffer</Td><Td>7.65</Td></Tr>
 
-
<Tr><Td>total</Td><Td>9μL</Td></Tr>
 
-
</Table>
 
<br>
<br>
-
 
+
<br>
-
 
+
①BP reaction<br>
-
<Table Border Cellspacing="0">
+
-
<Tr><Td></Td><Td>sample1</Td><Td>sample2</Td></Tr>
+
-
<Tr><Td>10ng/μL AIP2-MALT1</Td><Td>2.5μL</Td><Td>2.5μL</Td></Tr>
+
-
<Tr><Td>10×KOD plus buffer</Td><Td>5μL</Td><Td>5μL</Td></Tr>
+
-
<Tr><Td>2mM dNTPs</Td><Td>5μL</Td><Td>5μL</Td></Tr>
+
-
<Tr><Td>25mM MgSO<sub>4</sub></Td><Td>2μL</Td><Td>2.4μL</Td></Tr>
+
-
<Tr><Td>10P 5'primer</Td><Td>1.5μL</Td><Td>1.5μL</Td></Tr>
+
-
<Tr><Td>10P 3'primer</Td><Td>1.5μL</Td><Td>1.5μL</Td></Tr>
+
-
<Tr><Td>KOD plus</Td><Td>1μL</Td><Td>1μL</Td></Tr>
+
-
<Tr><Td>dH<sub>2</sub></Td><Td>31.5μL</Td><Td>31.1μL</Td></Tr>
+
-
<Tr><Td>total</Td><Td>50μL</Td><Td>50μL</Td></Tr>
+
-
</Table>
+
<br>
<br>
-
 
+
BP reaction was carried out in the following conditions.
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
-
<Tr><Td>temperature</Td><Td>time</Td><Td>cycle</Td></Tr>
+
<Tr><Td> attB TNFAIP3(80ng/μL) </Td><Td> 1μL </Td></Tr>
-
<Tr><Td>95°C</Td><Td>2min</Td><Td></Td></Tr>
+
<Tr><Td> pDONR(455ng/μL) </Td><Td> 0.35μL </Td></Tr>
-
<Tr><Td>95°C</Td><Td>15sec</Td><Td>35cycle</Td></Tr>
+
<Tr><Td> TE buffer </Td><Td> 7.65 </Td></Tr>
-
<Tr><Td>55°C</Td><Td>30sec</Td><Td>35cycle</Td></Tr>
+
<Tr><Td> total </Td><Td> 9μL </Td></Tr>
-
<Tr><Td>68°C</Td><Td>3min30sec</Td><Td>35cycle</Td></Tr>
+
-
<Tr><Td>68°C</Td><Td>3min30sec</Td><Td></Td></Tr>
+
-
<Tr><Td>14°C</Td><Td>∞</Td><Td></Td></Tr>
+
</Table>
</Table>
<br>
<br>
<br>
<br>
-
<img src="http://2012.igem.org/wiki/images/d/df/0803.png" width="500" height="300">
+
One uL of BP Clonase Ⅱ enzyme mix was added to this solution and incubated for 2.5 hours.<br>
-
<br><br>
+
<br>
 +
<br>
 +
② Transformation of E.coli by BP reaction products<br>
 +
2uL of BP TNFA reaction product was added to 100uL of E.coli XL1-Blue to do transform ation.<br>
 +
At last, cells were divided into 10,40,100,and 200 uL,and spreaded on LB Kanamycin(+)plate,Plats wera incubated for 16 hours at 37°C.<br>
 +
<br>
 +
<br>
 +
 
<h2>August 4th</h2>
<h2>August 4th</h2>
 +
<br><br>
-
 
+
Four independent 4 colonies were picked up from the plate incubated from 8/3 as described in ②,then cultured in 2.5mL of LB Kanamycin(+) liquid culture medium for 16 hours.<br>
 +
<br>
 +
<br>
<h2>August 5th</h2>
<h2>August 5th</h2>
 +
<br>
 +
<br>
-
<br><br>
+
Plasmid DNAs were isolated from E.coli cultured in 4 of LB liquid culture medium which we made on 8/4 by the alkaline-lysis method, we added the DNA to 20uL TE and added 10uL of the solution to 2uL of 6×Dye. Then we electrophoresed it with marker, 2uL of pDONR+ 2uL of 6×Dye+ 8uL of TE,in 0.7% agarose gel in following order (from left to right)<br>
-
<img src="http://2012.igem.org/wiki/images/3/31/0805.png" width="500" height="300">
+
<br>
-
<br><br>
+
Left control pDONR DNA, BP TNFA-1,-2,-3,-4<br>
-
<h2>August 6th</h2>
+
<br>
-
 
+
 +
<strong>Results</strong><br>
 +
<img src="http://2012.igem.org/wiki/images/3/31/0805.png" width="500" height="300"><br>
 +
<br>
 +
<br>
 +
<h2>August 6th</h2>
 +
<br>
 +
<br>
 +
E.coli carrying the plasmid sample -1 and -2 in LB Kanamycin(+)liquid culture medium for 16 hours.<br>
 +
<br>
 +
<br>
<h2>August 7th</h2>
<h2>August 7th</h2>
-
<br><br>
+
<br>
-
<img src="http://2012.igem.org/wiki/images/2/2e/0807.png" width="500" height="300">
+
<br>
-
<br><br>
+
①Purification of candidate pENTR-TNFAIP3<br>
-
 
+
We purified candidate plasmid-1 and -2 by QIA Prep Spin Miniprep Kit, dissolved  it in 30uL of Buffer EB.<br>
 +
Then,the samples were electrophoresed in 0.7% agarose gel.<br>
 +
<br>
 +
We made samples as follows.
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
-
<Tr><Td></Td><Td>pDONR</Td><Td>BP TNFA-1</Td><Td>BP TNFA-2</Td></Tr>
+
<Tr><Td>  </Td><Td> pDONR </Td><Td> BP TNFA-1 </Td><Td> BP TNFA-2 </Td></Tr>
-
<Tr><Td>DNA sample</Td><Td>1μL</Td><Td>1μL</Td><Td>1μL</Td></Tr>
+
<Tr><Td> DNA sample </Td><Td> 1μL </Td><Td> 1μL </Td><Td> 1μL </Td></Tr>
-
<Tr><Td>6×Dye</Td><Td>1μL</Td><Td>1μL</Td><Td>1μL</Td></Tr>
+
<Tr><Td> 6×Dye </Td><Td> 1μL </Td><Td> 1μL </Td><Td> 1μL </Td></Tr>
-
<Tr><Td>dH<sub>2</sub>O</Td><Td>4μL</Td><Td>4μL</Td><Td>4μL</Td></Tr>
+
<Tr><Td> dH<sub>2</sub>O </Td><Td> 4μL </Td><Td> 4μL </Td><Td> 4μL </Td></Tr>
-
<Tr><Td>total</Td><Td>6μL</Td><Td>6μL</Td><Td>6μL</Td></Tr>
+
<Tr><Td> total </Td><Td> 6μL </Td><Td> 6μL </Td><Td> 6μL </Td></Tr>
</Table>
</Table>
<br>
<br>
-
 
+
<br>
 +
We electrophoresed them in following order.<br>
 +
Left marker 5uL control pDONR DNA ,pENTR-TNFAIP3-1 DNA,pENTR-TNFAIP3 -2 DNA marker 4uL marker 3uL Wright.<br>
 +
<br>
 +
<strong>Results</strong><br>
 +
<img src="http://2012.igem.org/wiki/images/2/2e/0807.png" width="500" height="300">
 +
<br>
 +
Concentration of pENTR-TNFAIP3-2 DNA was estimated at around 100ng/uL.<br>
 +
<br>
 +
<br>
 +
② LR reaction<br>
 +
We made following solution(vials)in 1.5mL tube.<br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
-
<Tr><Td>BP TNFA-2</Td><Td>2μL</Td></Tr>
+
<Tr><Td> BP TNFA-2 </Td><Td> 2μL </Td></Tr>
-
<Tr><Td>pTFW(Destination vector)</Td><Td>0.5μL</Td></Tr>
+
<Tr><Td> pTFW(Destination vector) </Td><Td> 0.5μL </Td></Tr>
-
<Tr><Td>TE buffer</Td><Td>6.5μL</Td></Tr>
+
<Tr><Td> TE buffer </Td><Td> 6.5μL </Td></Tr>
-
<Tr><Td>total</Td><Td>9μL</Td></Tr>
+
<Tr><Td> total </Td><Td> 9μL </Td></Tr>
</Table>
</Table>
 +
<br>
 +
we added 1uL of LR clonaseⅡ enzyme mix to this solution and incubate it for 2.5 hours.<br>
 +
<br>
 +
③ Transformation of E.coli with LR reaction products.<br>
 +
Transformation of E.coli was carried out by adding 2uL of LR reaction products to 100uL of XL1-Blue and plated on the LB ampicillin(+)plate and cultured at 37°C.<br>
 +
<br>
<br>
<br>
 +
 +
WEEK1終わり
WEEK1終わり
-
<br>
 
 +
<br>
-
<h2>August 8th</h2>
 
-
<head>
+
 
 +
<h2>August 8th</h2>
 +
<br>
 +
<br>
 +
We isolated 5 colonies from the LB plate, and cultured.<br>
 +
<br>
 +
<br>
 +
 
<h2>August 9th</h2>
<h2>August 9th</h2>
 +
<br>
 +
<br>
 +
①Purification of the candidate pUAS-flag-TNFAIP3 DNA<br>
 +
<br>
 +
The candidate pUAS-flag-TNFAIP3 DNAs from five independent sample -1,-2,-3,-4 and -5 were purified by the alkaline-lysis method.<br>
 +
We added 0.2uL of 10ng/uL RNase A to them and incubate them for 1 hour.<br>
 +
<br>
 +
<br>
 +
②Characterization of the candidate pUAS-flag-TNFAIP3 DNA<br>
 +
<br>
 +
The candidate pUAS-flag-TNFAIP3 DNA was digested wit EcoRⅠ as follows.<br>
 +
<br> 
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
-
<Tr><Td>DNA sample</Td><Td>1μL</Td></Tr>
+
<Tr><Td> Each diluted DNA sample </Td><Td> 1μL </Td></Tr>
-
<Tr><Td>10×H buffer</Td><Td>0.5μL</Td></Tr>
+
<Tr><Td> 10×H buffer </Td><Td> 0.5μL </Td></Tr>
-
<Tr><Td>EcoRⅠ</Td><Td>0.2μL</Td></Tr>
+
<Tr><Td> EcoRⅠ </Td><Td> 0.2μL </Td></Tr>
-
<Tr><Td>dH<sub>2</sub>O</Td><Td>3.3μL</Td></Tr>
+
<Tr><Td> dH<sub>2</sub>O </Td><Td> 3.3μL </Td></Tr>
-
<Tr><Td>total</Td><Td>5μL</Td></Tr>
+
<Tr><Td> total </Td><Td> 5μL </Td></Tr>
</Table>
</Table>
<br>
<br>
<br>
<br>
 +
 +
After incubation of these samples for 1hour, we added 1uL of 6×Dye to each of them and electrophoresed in 0.7% agarose gel in following order.<br>
 +
Left, 10fold dilution, 2uL marker, 20fold dilution, 30fold dilution, 1uL marker, 40fold dilution, 0.5uL marker right.<br>
 +
<br>
 +
<br>
 +
 +
 +
<strong>Results</strong><br>
<img src="http://2012.igem.org/wiki/images/9/9b/0809.png" width="500" height="300">
<img src="http://2012.igem.org/wiki/images/9/9b/0809.png" width="500" height="300">
<br><br>
<br><br>
 +
The concerntration of the candidate pUAS-flag-TNFAIP3 DNA was estimated as 200ng/uL.<br>
 +
<br>
 +
<br>
 +
③ PCR amplification of the insert DNA<br>
 +
<br>
 +
The four 5'primers were designed for PCR as follows.<br>
 +
<br>
-
<Table Border Cellspacing="0">
+
・GW3r<br>
-
<Tr><Td></Td><Td>1-2</Td></Tr>
+
5’-ATCGAGGCCTGTCTAGAGAAGC<br>
-
<Tr><Td>50ng/μL LR TNFA-1</Td><Td>1μL</Td></Tr>
+
・TNFA-1<br>
-
<Tr><Td>10×rTaq buffer</Td><Td>2μL</Td></Tr>
+
5’-GGCTTTGCTATGATACTCGGAACTG<br>
-
<Tr><Td>2mM dNTPs</Td><Td>2μL</Td></Tr>
+
・TNFA-2<br>
-
<Tr><Td>25mM MgCl<sub>2</sub></Td><Td>0.8μL</Td></Tr>
+
5’-GTAAAATGTGAAACGCCCAACTGC<br>
-
<Tr><Td>10P 5'primer</Td><Td>0.6μL</Td></Tr>
+
・TNFA-3<br>
-
<Tr><Td>10P 3'primer</Td><Td>0.6μL</Td></Tr>
+
5’-GGACTCCAGAAAACAAGGGCTTT<br>
-
<Tr><Td>rTaq</Td><Td>0.4μL</Td></Tr>
+
<br>
-
<Tr><Td>dH<sub>2</sub>O</Td><Td>12.6μL</Td></Tr>
+
<br>
-
<Tr><Td>total</Td><Td>20μL</Td></Tr>
+
 
-
</Table>
+
 
 +
The 3’ primer was designed as follows.<br>
 +
・SVr<br>
 +
5’-GGCATTCCACCACTGCTCCC<br>
 +
<br>
 +
<br>
 +
 
 +
 
 +
PCR reactions with the following combinations of primers were carried out.<br>
 +
sample1→GW3r―SVr<br>
 +
sample2→TNFA-1―SVr<br>
 +
sample3→TNFA-2―SVr<br>
 +
sample4→TNFA-3―SVr<br>
 +
 
 +
<br>
<br>
<br>
 +
PCR was carried out in the following reactions.
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
-
<Tr><Td>temperature</Td><Td>time</Td><Td>cycle</Td></Tr>
+
<Tr><Td>  </Td><Td> Each sample </Td></Tr>
-
<Tr><Td>95°C</Td><Td>2min</Td><Td></Td></Tr>
+
<Tr><Td> 50ng/μL LR TNFA-1 </Td><Td> 1μL </Td></Tr>
-
<Tr><Td>95°C</Td><Td>15sec</Td><Td>25cycle</Td></Tr>
+
<Tr><Td> 10×rTaq buffer </Td><Td> 2μL </Td></Tr>
-
<Tr><Td>55°C</Td><Td>30sec</Td><Td>25cycle</Td></Tr>
+
<Tr><Td> 2mM dNTPs </Td><Td> 2μL </Td></Tr>
-
<Tr><Td>68°C</Td><Td>2min30sec</Td><Td>25cycle</Td></Tr>
+
<Tr><Td> 25mM MgCl<sub>2</sub> </Td><Td> 0.8μL </Td></Tr>
-
<Tr><Td>68°C</Td><Td>2min30sec</Td><Td></Td></Tr>
+
<Tr><Td> 10P 5'primer </Td><Td> 0.6μL </Td></Tr>
-
<Tr><Td>14°C</Td><Td></Td><Td></Td></Tr>
+
<Tr><Td> 10P 3'primer </Td><Td> 0.6μL </Td></Tr>
 +
<Tr><Td> rTaq </Td><Td> 0.4μL </Td></Tr>
 +
<Tr><Td> dH<sub>2</sub>O </Td><Td> 12.6μL </Td></Tr>
 +
<Tr><Td> total </Td><Td> 20μL </Td></Tr>
 +
</Table>
 +
<br><br>
 +
 
 +
Reaction conditions of PCR
 +
<Table Border Cellspacing="0">
 +
<Tr><Td> temperature </Td><Td> time </Td><Td> cycle </Td></Tr>
 +
<Tr><Td> 95°C </Td><Td> 2min </Td><Td>  </Td></Tr>
 +
<Tr><Td> 95°C </Td><Td> 15sec </Td><Td> 25cycle </Td></Tr>
 +
<Tr><Td> 55°C </Td><Td> 30sec </Td><Td> 25cycle </Td></Tr>
 +
<Tr><Td> 68°C </Td><Td> 2min30sec </Td><Td> 25cycle </Td></Tr>
 +
<Tr><Td> 68°C </Td><Td> 2min30sec </Td><Td>  </Td></Tr>
 +
<Tr><Td> 14°C </Td><Td> ∞ </Td><Td>  </Td></Tr>
</Table>
</Table>
<br>
<br>
Line 249: Line 893:
<h2>August 10th</h2>
<h2>August 10th</h2>
 +
<br>
 +
<br>
 +
 +
Electrophoresis of PCR products was carried out.<br>
 +
After adding 2uL of 6×Dye to each 10uL of sample, we electrophoresed them in 0.7% agarose gel in following order.<br>
 +
<br>
 +
Left marker5uL sample1 sample2 sample3 sample4 Wright<br>
 +
<br>
 +
 +
<strong>Results</strong><br>
-
<br><br>
 
<img src="http://2012.igem.org/wiki/images/8/84/0810.png" width="500" height="300">
<img src="http://2012.igem.org/wiki/images/8/84/0810.png" width="500" height="300">
-
<br><br>
+
<br>
 +
The sizes of the PCR products were unexpected ones. We therefore concluded the construction of pUAS-flag-TNFAIP3 DNA was not successful.<br>
 +
<br>
 +
<br>
 +
 
 +
 
 +
 
 +
 
 +
 
<h2>August 13th</h2>
<h2>August 13th</h2>
 +
<br>
 +
<br>
 +
Cut check of the plasmid DNA(the candidate pENTR-TNFAIP3) after BP reaction.<br>
 +
<br>
 +
The candidate pENTR-TNFAIP3 was digested with HindⅢ that will cut out the insert DNA.<br>
 +
<br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
-
<Tr><Td>1-1</Td><Td>1μL</Td></Tr>
+
<Tr><Td> the candidate pENTR-TNFAIP3 prepared on 8/6 </Td><Td> 1μL </Td></Tr>
-
<Tr><Td>10×M buffer</Td><Td>0.5μL</Td></Tr>
+
<Tr><Td> 10×M buffer </Td><Td> 0.5μL </Td></Tr>
-
<Tr><Td>Hind Ⅲ</Td><Td>0.2μL</Td></Tr>
+
<Tr><Td> Hind Ⅲ </Td><Td> 0.2μL </Td></Tr>
-
<Tr><Td>dH<sub>2</sub>O</Td><Td>3.3μL</Td></Tr>
+
<Tr><Td> dH<sub>2</sub>O </Td><Td> 3.3μL </Td></Tr>
-
<Tr><Td>total</Td><Td>5μL</Td></Tr>
+
<Tr><Td> total </Td><Td> 5μL </Td></Tr>
</Table>
</Table>
<br>
<br>
 +
<br>After reaction, samples were electrophoresed in following order.<br>
 +
<br>
 +
Left: 1kb marker5uL cut sample <br>
 +
Right: uncut sample<br>
 +
<br>
 +
<br>
 +
<strong>Results</strong>
<br>
<br>
<img src="http://2012.igem.org/wiki/images/2/2a/0813.png" width="500" height="300">
<img src="http://2012.igem.org/wiki/images/2/2a/0813.png" width="500" height="300">
<br><br>
<br><br>
 +
Insert size was different from the expected one. We therefore concluded that the construction of entry DNA was not successful.<br>
 +
<br>
 +
<br>
<h2>August 14th</h2>
<h2>August 14th</h2>
 +
<br>
 +
<br>
 +
Based on the results, we decided to carry out the PCR reaction for TNFAIP3 DNA again.<br>
 +
<br>
-
 
+
Composition of the reaction
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
-
<Tr><Td>1-1</Td><Td>sample1</Td></Tr>
+
<Tr><Td>  </Td><Td> sample1 </Td></Tr>
-
<Tr><Td>10ng/μL TNFAIP3</Td><Td>6μL</Td></Tr>
+
<Tr><Td> 10ng/μL TNFAIP3 </Td><Td> 6μL </Td></Tr>
-
<Tr><Td>10×KOD plus buffer</Td><Td>10μL</Td></Tr>
+
<Tr><Td> 10×KOD plus buffer </Td><Td> 10μL </Td></Tr>
-
<Tr><Td>2mM dNTPs</Td><Td>10μL</Td></Tr>
+
<Tr><Td> 2mM dNTPs </Td><Td> 10μL </Td></Tr>
-
<Tr><Td>25mM MgSO<sub>4</sub></Td><Td>3.2μL</Td></Tr>
+
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 3.2μL </Td></Tr>
-
<Tr><Td>10P 5'primer</Td><Td>3μL</Td></Tr>
+
<Tr><Td> 10P 5'primer </Td><Td> 3μL </Td></Tr>
-
<Tr><Td>10P 3'primer</Td><Td>3μL</Td></Tr>
+
<Tr><Td> 10P 3'primer </Td><Td> 3μL </Td></Tr>
-
<Tr><Td>KOD plus</Td><Td>2μL</Td></Tr>
+
<Tr><Td> KOD plus </Td><Td> 2μL </Td></Tr>
-
<Tr><Td>dH<sub>2</sub>O</Td><Td>62.8μL</Td></Tr>
+
<Tr><Td> dH<sub>2</sub>O </Td><Td> 62.8μL </Td></Tr>
-
<Tr><Td>total</Td><Td>100μL</Td></Tr>
+
<Tr><Td> total </Td><Td> 100μL </Td></Tr>
</Table>
</Table>
 +
<br>
<br>
<br>
-
 
+
Reaction conditions
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
-
<Tr><Td>temperature</Td><Td>time</Td><Td>cycle</Td></Tr>
+
<Tr><Td> temperature </Td><Td> time </Td><Td> cycle </Td></Tr>
-
<Tr><Td>95°C</Td><Td>2min</Td><Td></Td></Tr>
+
<Tr><Td> 95°C </Td><Td> 2min </Td><Td>  </Td></Tr>
-
<Tr><Td>95°C</Td><Td>15sec</Td><Td>25cycle</Td></Tr>
+
<Tr><Td> 95°C </Td><Td> 15sec </Td><Td> 25cycle </Td></Tr>
-
<Tr><Td>60°C</Td><Td>30sec</Td><Td>25cycle</Td></Tr>
+
<Tr><Td> 60°C </Td><Td> 30sec </Td><Td> 25cycle </Td></Tr>
-
<Tr><Td>68°C</Td><Td>2min30sec</Td><Td>25cycle</Td></Tr>
+
<Tr><Td> 68°C </Td><Td> 2min30sec </Td><Td> 25cycle </Td></Tr>
-
<Tr><Td>68°C</Td><Td>2min30sec</Td><Td></Td></Tr>
+
<Tr><Td> 68°C </Td><Td> 2min30sec </Td><Td>  </Td></Tr>
-
<Tr><Td>14°C</Td><Td></Td><Td></Td></Tr>
+
<Tr><Td> 14°C </Td><Td> ∞ </Td><Td>  </Td></Tr>
</Table>
</Table>
<br>
<br>
<br>
<br>
 +
PCR product was applied to the 0.7% agarose gel electrophoresis.<br>
 +
<br>
 +
 +
<strong>Results</strong><br>
<img src="http://2012.igem.org/wiki/images/c/c4/0814.png" width="500" height="300">
<img src="http://2012.igem.org/wiki/images/c/c4/0814.png" width="500" height="300">
-
<br><br>
+
<br>
 +
<br>
 +
<br>
WEEK2終わり
WEEK2終わり
<br>
<br>
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
Line 316: Line 1,014:
<br>
<br>
<h2>August 20th</h2>
<h2>August 20th</h2>
-
<br>
+
<br><br>
Line 323: Line 1,021:
Composition  
Composition  
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
-
<Tr><Td></Td><Td>a product of PCR attB TNFAIP3(8/1) </Td></Tr>
+
<Tr><Td>  </Td><Td> a product of PCR attB TNFAIP3(8/1) </Td></Tr>
-
<Tr><Td>DNA sample</Td><Td>90μL</Td></Tr>
+
<Tr><Td> DNA sample </Td><Td> 90μL </Td></Tr>
-
<Tr><Td>6×Dye</Td><Td>18μL</Td></Tr>
+
<Tr><Td> 6×Dye </Td><Td> 18μL </Td></Tr>
-
<Tr><Td>Total</Td><Td>108μL</Td></Tr>
+
<Tr><Td> Total </Td><Td> 108μL </Td></Tr>
</Table>
</Table>
<br>
<br>
Line 339: Line 1,037:
<h2>August 21st</h2>
<h2>August 21st</h2>
<br>
<br>
 +
<strong>TNFA and API2</strong>
 +
<br><br>
Line 344: Line 1,044:
  We did agarose gel electrophoresis (0.7% gel). (1kb marker-3uL, 5times dilution attB TNFAIP3-1uL (make August 20 ), 10times dilution-1uL, 1kb marker-2uL, 15times dilution-1uL, 20times dilution-1uL, 1kb marker-1uL )<br><br>
  We did agarose gel electrophoresis (0.7% gel). (1kb marker-3uL, 5times dilution attB TNFAIP3-1uL (make August 20 ), 10times dilution-1uL, 1kb marker-2uL, 15times dilution-1uL, 20times dilution-1uL, 1kb marker-1uL )<br><br>
-
<strong>Results</strong>  
+
<strong>Results</strong> <br>
  We estimated attB TNFAIP3(we make this time) is 35ng/uL<br><br>
  We estimated attB TNFAIP3(we make this time) is 35ng/uL<br><br>
 +
 +
<br>
 +
<img src="http://2012.igem.org/wiki/images/6/67/0821kit.png" width="500" height="300">
 +
<br>
2. BP reaction<br>
2. BP reaction<br>
We adjusted solution (vials) on 1.5mL tube to next composition.<br>
We adjusted solution (vials) on 1.5mL tube to next composition.<br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
-
<Tr><Td>attB TNFAIP3(35ng/μL)</Td><Td>2μL</Td></Tr>
+
<Tr><Td> attB TNFAIP3(35ng/μL) </Td><Td> 2μL </Td></Tr>
-
<Tr><Td>pDONR(150ng/μL)</Td><Td>1μL</Td></Tr>
+
<Tr><Td> pDONR(150ng/μL) </Td><Td> 1μL </Td></Tr>
-
<Tr><Td>TE buffer</Td><Td>5μL</Td></Tr>
+
<Tr><Td> TE buffer </Td><Td> 5μL </Td></Tr>
-
<Tr><Td>Total</Td><Td>8μL</Td></Tr>
+
<Tr><Td> Total </Td><Td> 8μL </Td></Tr>
</Table>
</Table>
<br>
<br>
Line 369: Line 1,073:
<h2>August 22nd</h2>
<h2>August 22nd</h2>
<br>
<br>
-
 
+
<strong>TNFA and API2</strong>
 +
<br><br>
We separated six colonies from 40uL spread plate of BP TNFAIP3 (make August 21 ) .<br>
We separated six colonies from 40uL spread plate of BP TNFAIP3 (make August 21 ) .<br>
We cultivated these colonies on 2.5mL Kanamycin(+) LB culture medium and we kept shaking this medium overnight.<br>
We cultivated these colonies on 2.5mL Kanamycin(+) LB culture medium and we kept shaking this medium overnight.<br>
Line 376: Line 1,081:
</div>
</div>
</html>
</html>
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 +
 +
 +
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<UL>
 +
<LI><P STYLE="margin-bottom: 0cm; page-break-before: always"><FONT FACE=""><B>
 +
</B></FONT><FONT FACE=""><SPAN LANG="en-US"><B>DNA purification and
 +
precipitation</B></SPAN></FONT></P>
 +
</UL>

Latest revision as of 01:55, 26 September 2013

Template:KIT-Kyoto-1 Template:KIT-Kyoto-header

Protocol


Miniprep

Solution Ⅰ (50 mM Tris-HCl and 10 mM EDTA, and 50 µg/mL RNase A, pH 8.0 (25˚C))

Solution Ⅱ (0.2 M NaOH and 1 % SDS)

Solution Ⅲ (4 M guanidine hydrochloride and 0.5 M potassium acetate, pH 4.2)

Solution Ⅳ (5 M guanidine hydrochloride, 20 mM Tris-HCl, and 38% ethanol, pH 6.6 (25˚C))

Solution Ⅴ (2 mM Tris-HCl and 80% ethanol, pH 7.5 (25˚C))


Pick up a single colony from plate and cultivate it overnight in 3ml LB medium containing appropriate antibiotic at 37˚C˚.

Transfer the culture into a 1.5ml tube, centrifuge at 13,000 rpm for 1 minute, and discard the supernatant (2 times).

Add 250 µL of SolutionⅠ to the pellet and mix well with vortex.

Add 250 µL of SolutionⅡ and mix well by turning the tube upside down several times.

Add 350 µL of SolutionⅢ and by turning the tube upside down several times.

Centrifuge at 13,000 rpm for 5 minutes.

Transfer the supernatant (approx. 850µL) to a mini prep column.

Centrifuge at 13,000 rpm for 1 minute and discard the flow-through.

Add 500 µL of SolutionⅣ.

Centrifuge at 13,000 rpm for 1 minute and discard the flow-through.

Add 700 µL of SolutionⅤ.

Centrifuge at 13,000 rpm for 1 minute and discard the flow-through (2 times).

Set the column on a new 1.5ml tube and add 100 µL of nuclease-free water.

Centrifuge at 13,000 rpm for 1 minute and collect plasmid DNA.



Rapid check of the insert by colony cracking

Add 45 µL of cracking solution(3% w/v SDS ,50mM Tris -base ,pH12.6) into each tube.

Suspend a small quantity of the colony in a tube using a sterilized toothpick.

Incubate the tube at 65°C for 10 minutes.

Add a drop of 10x Loading Buffer.

Add equal volume of phenol/chloroform.

Mix with vortex.

Centrifuge at 13,000rpm for 3 minutes.

Apply the supernatant to 1% agarose gel electrophoresis.

Stain the gel in ethidium bromide solution for 10 minutes.

Plasmid DNA can be detected by UV-illuminator as a band between genomic DNA band and low molecular size RNAs.


  • DNA purification and precipitation


  1. Mix 350 µL of sterilized water and 50 µL of 3 M sodium acetate with DNA sample.

  2. Mix DNA solution with equal volume of phenol/chloroform by vortex.

  3. Centrifuge at 13,000 rpm for 5 min, then transfer the supernatant into a new tube.

  4. Add equal volume of 2-propanol, and mix by turning the tube upside down.

  5. Centrifuge at 14,500 rpm for 10 min at 4˚C.

  6. Carefully decant the supernatant.

  7. Add adequate volume of 70 % ethanol.

  8. Centrifuge at 14,500 rpm for 5 min at 4˚C.

  9. Carefully decant the supernatant.

  10. Dry in the desiccator under vacuum for 5 min.

  11. You can get dried DNA pellet.



  • PCR


  1. Adjust the concentration of each primer to 100 pmol/µL with sterilized water.

  2. Mix 10µL of forward and reverse primer solutions with 80 µL H2O in a new tube (final primer concentration is 10 pmol/µL).

  3. Use 1 µL of primer mix for PCR.

Recipe for PRC is as follows:

Buffer

50 µL

dNTP

20 µL

Primer mix

1 µL

DNA sample

0.5 µL

KOD-FX

2 µL

H2O

26.5 µL

total

100 µL


KOD-FX(Toyobo)



SDS polyacrylamide gel electrophoresis (SDS-PAGE)


12.5% separation gel (recipe for a sheet of gel)

Mili Q water 2.59 ml

acrylamide solution(30 %) 3.33 ml

0.5M Tris (pH8.8) 2 ml

10%SDS 80 µL

10%APS 27 µL

TEMED 4 µL

Apply the acrylamide solution mix to the PAGE glass plate.

Deposit H2O carefully on the top of the acrylamide solution mix.

Wait for 10 min for polymerization.


Stacking gel

Mili Q water 2.89 ml

Acrylamide 0.79 ml

0.5M Tris (pH 6.8) 1.25 ml

10% SDS 50 µL

10%APS 17 µL

TEMED 5 µL

After the polymerization of the separation gel, remove the H2O of the top layer.

Apply stacking gel mix on the running gel and put the comb to make wells.

After the stacking gel has fully polymerized, remove the comb and rinse the top of the gel with H2O and then remove H2O.


Sample preparation

Collect bacterial cells.

Add 450ul of H2O and 50 µL of FastBreak Cell Lysis Reagent (Madison, Wisconsin, USA Promega).

Mix them for 15minutes with shaker.

Add 100ul of 6SDS-Sample buffer and mix with vortex.

Heat samples in a heating block at 99°C for 5 minutes.


Running samples

Set the gel on the electrophoresis apparatus and apply SDS running buffer (Tris 25mM,Glysine 191mM,SDS0.1%)

Apply the samples and markers.

Operate at constant current (25mA) for 65 minutes per sheet of gel.


Staining

Place the gel in stacking solution( 50%ethanol,10%acetic acid) and shake gently it for 5 minutes.

Remove the stacking solution and add 100ml of staining solution (0.25% CBB R250、5%methanol、7.5%acetic acid).

Warm the gel for 1 minute with a microwave (500W).

Remove the staining solution and rinse the gel with water.


  • Isolation and purification of DNA bands


  1. Clip DNA band from agarose gel and transfer it into a 1.5 ml tube.

  2. Add H2O. Melt the gel at 65˚C in a heating block.

  3. Add equal volume of phenol. Mix well with vortex.

  4. Centrifuge at 13,000 rpm for 5 min.

  5. Transfer the supernatant into a new tube and mix with equal volume of phenol/chloroform.

  6. Perform the same method of DNA precipitation using 2-propanol.


    Blue gel
    ・50×TAE 100ml
    ・Agarose(nacalai tesque) 1.0g
    ・Gel indicator(Biodynamics laboratory) 200ul





<Ligation>


Dissolve the purified and dried insert DNA in 5µL of H2O.

Dissolve the purified and dried vector DNA in 5µL of H2O.

Mix the two solutions.

Add 10µL of Ligation Mix (Wako) to it.

Incubate for 15 minutes at room temperature.



<Transformation>


Add the ligation solution to competent cells (in a clean bench).

Place tubes on ice for 20 minutes.

Heat shock treatment at 42°C for 35 seconds.

Place tubes on ice for 2 minutes.

Add 1mL of LB medium into the tube (in a clean bench).

Incubate the samples for 20 minutes at 37°C.

Centrifuge at 7,000rpm for 3 minutes.

Discard the most of supernatant, and spread the remaining cells and LD medium in the tube on the plates (in a clean bench).

Incubate plates overnight at 37°C.























August 1st



Since it takes at least one month to establish transgenic flies, we decided to first construct the P-element plasmid for microinjection into Drosophia embryos.

① We performed PCR in the following conditions.

(ⅰ)TNFAIP3
Composition for reaction
   sample1 
 10ng/μL TNFAIP3  6μL 
 10× KOD plus buffer  10μL 
 2mM dNTPs  10μL 
 25mM MgSO4  3.2μL 
 10P 5'primer  3μL 
 10P 3'primer  3μL 
 KOD plus  2μL 
 dH2O  62.8μL 
 Total  100μL 

Reaction conditions
 temperature  time  cycle 
 95°C  2min.   
 95°C  15sec  25cycle 
 60°C  30sec  25cycle 
 68°C  2min30sec  25cycle 
 68°C  2min30sec   
 14°C  ∞   


August 2nd



① PCR product check

We run 0.7% agarose gel electorophoresis in the following conditions. Composition
   8/1 the attB TNFAIP3 PCR products 
 DNA sample  10μL 
 6×Dye  2μL 
 total  12μL 


The order of sample application
Left : 1kb marker(5uL)
Right: attB TNFAIP3

Results


② DNA purification from gel
We electrophoresed in 0.7% agarose gel in the following conditions.

Composition
   8/1 attB TNFAIP3 PCRproduct 
 DNA sample  90μL 
 6×Dye  18μL 
 total  108μL 


We applied half of 108 uL of sample to each of two lanes of Agaroe gel with 5uL of 1kb marker.Then we detected the DNA band in the gel.

Results

We purifed DNA from the gel by QIA Quick Gel Extraction Kit.


・Eatimation of the amount of attB TNFAIP3 DNA fragments by electrophoresis.
Order of samples applied to the agarose gel
The samples were electrophoresed in 0.7% agarose gel in following order.
Right to left:3uL of 1kb marker 1uL of 5 fold dilution of attB TNFAIP3 DNA fragments,1uL of 10 fold dilution of attB TNFAIP3 DNA fragments, 1uL of 20 fold dilution of attB TNFAIP3 DNA fragments, 1uL of 1kb marker.

Results

We have estimated amount of the attB TNFAIP3 DNA to be 80ng/uL.


August 3rd



①BP reaction

BP reaction was carried out in the following conditions.
 attB TNFAIP3(80ng/μL)  1μL 
 pDONR(455ng/μL)  0.35μL 
 TE buffer  7.65 
 total  9μL 


One uL of BP Clonase Ⅱ enzyme mix was added to this solution and incubated for 2.5 hours.


② Transformation of E.coli by BP reaction products
2uL of BP TNFA reaction product was added to 100uL of E.coli XL1-Blue to do transform ation.
At last, cells were divided into 10,40,100,and 200 uL,and spreaded on LB Kanamycin(+)plate,Plats wera incubated for 16 hours at 37°C.


August 4th



Four independent 4 colonies were picked up from the plate incubated from 8/3 as described in ②,then cultured in 2.5mL of LB Kanamycin(+) liquid culture medium for 16 hours.


August 5th



Plasmid DNAs were isolated from E.coli cultured in 4 of LB liquid culture medium which we made on 8/4 by the alkaline-lysis method, we added the DNA to 20uL TE and added 10uL of the solution to 2uL of 6×Dye. Then we electrophoresed it with marker, 2uL of pDONR+ 2uL of 6×Dye+ 8uL of TE,in 0.7% agarose gel in following order (from left to right)

Left control pDONR DNA, BP TNFA-1,-2,-3,-4

Results



August 6th



E.coli carrying the plasmid sample -1 and -2 in LB Kanamycin(+)liquid culture medium for 16 hours.


August 7th



①Purification of candidate pENTR-TNFAIP3
We purified candidate plasmid-1 and -2 by QIA Prep Spin Miniprep Kit, dissolved it in 30uL of Buffer EB.
Then,the samples were electrophoresed in 0.7% agarose gel.

We made samples as follows.
   pDONR  BP TNFA-1  BP TNFA-2 
 DNA sample  1μL  1μL  1μL 
 6×Dye  1μL  1μL  1μL 
 dH2O  4μL  4μL  4μL 
 total  6μL  6μL  6μL 


We electrophoresed them in following order.
Left marker 5uL control pDONR DNA ,pENTR-TNFAIP3-1 DNA,pENTR-TNFAIP3 -2 DNA marker 4uL marker 3uL Wright.

Results

Concentration of pENTR-TNFAIP3-2 DNA was estimated at around 100ng/uL.


② LR reaction
We made following solution(vials)in 1.5mL tube.
 BP TNFA-2  2μL 
 pTFW(Destination vector)  0.5μL 
 TE buffer  6.5μL 
 total  9μL 

we added 1uL of LR clonaseⅡ enzyme mix to this solution and incubate it for 2.5 hours.

③ Transformation of E.coli with LR reaction products.
Transformation of E.coli was carried out by adding 2uL of LR reaction products to 100uL of XL1-Blue and plated on the LB ampicillin(+)plate and cultured at 37°C.


WEEK1終わり

August 8th



We isolated 5 colonies from the LB plate, and cultured.


August 9th



①Purification of the candidate pUAS-flag-TNFAIP3 DNA

The candidate pUAS-flag-TNFAIP3 DNAs from five independent sample -1,-2,-3,-4 and -5 were purified by the alkaline-lysis method.
We added 0.2uL of 10ng/uL RNase A to them and incubate them for 1 hour.


②Characterization of the candidate pUAS-flag-TNFAIP3 DNA

The candidate pUAS-flag-TNFAIP3 DNA was digested wit EcoRⅠ as follows.

 Each diluted DNA sample  1μL 
 10×H buffer  0.5μL 
 EcoRⅠ  0.2μL 
 dH2O  3.3μL 
 total  5μL 


After incubation of these samples for 1hour, we added 1uL of 6×Dye to each of them and electrophoresed in 0.7% agarose gel in following order.
Left, 10fold dilution, 2uL marker, 20fold dilution, 30fold dilution, 1uL marker, 40fold dilution, 0.5uL marker right.


Results


The concerntration of the candidate pUAS-flag-TNFAIP3 DNA was estimated as 200ng/uL.


③ PCR amplification of the insert DNA

The four 5'primers were designed for PCR as follows.

・GW3r
5’-ATCGAGGCCTGTCTAGAGAAGC
・TNFA-1
5’-GGCTTTGCTATGATACTCGGAACTG
・TNFA-2
5’-GTAAAATGTGAAACGCCCAACTGC
・TNFA-3
5’-GGACTCCAGAAAACAAGGGCTTT


The 3’ primer was designed as follows.
・SVr
5’-GGCATTCCACCACTGCTCCC


PCR reactions with the following combinations of primers were carried out.
sample1→GW3r―SVr
sample2→TNFA-1―SVr
sample3→TNFA-2―SVr
sample4→TNFA-3―SVr


PCR was carried out in the following reactions.
   Each sample 
 50ng/μL LR TNFA-1  1μL 
 10×rTaq buffer  2μL 
 2mM dNTPs  2μL 
 25mM MgCl2  0.8μL 
 10P 5'primer  0.6μL 
 10P 3'primer  0.6μL 
 rTaq  0.4μL 
 dH2O  12.6μL 
 total  20μL 


Reaction conditions of PCR
 temperature  time  cycle 
 95°C  2min   
 95°C  15sec  25cycle 
 55°C  30sec  25cycle 
 68°C  2min30sec  25cycle 
 68°C  2min30sec   
 14°C  ∞   

August 10th



Electrophoresis of PCR products was carried out.
After adding 2uL of 6×Dye to each 10uL of sample, we electrophoresed them in 0.7% agarose gel in following order.

Left marker5uL sample1 sample2 sample3 sample4 Wright

Results

The sizes of the PCR products were unexpected ones. We therefore concluded the construction of pUAS-flag-TNFAIP3 DNA was not successful.


August 13th



Cut check of the plasmid DNA(the candidate pENTR-TNFAIP3) after BP reaction.

The candidate pENTR-TNFAIP3 was digested with HindⅢ that will cut out the insert DNA.

 the candidate pENTR-TNFAIP3 prepared on 8/6  1μL 
 10×M buffer  0.5μL 
 Hind Ⅲ  0.2μL 
 dH2O  3.3μL 
 total  5μL 


After reaction, samples were electrophoresed in following order.

Left: 1kb marker5uL cut sample
Right: uncut sample


Results


Insert size was different from the expected one. We therefore concluded that the construction of entry DNA was not successful.


August 14th



Based on the results, we decided to carry out the PCR reaction for TNFAIP3 DNA again.

Composition of the reaction
   sample1 
 10ng/μL TNFAIP3  6μL 
 10×KOD plus buffer  10μL 
 2mM dNTPs  10μL 
 25mM MgSO4  3.2μL 
 10P 5'primer  3μL 
 10P 3'primer  3μL 
 KOD plus  2μL 
 dH2O  62.8μL 
 total  100μL 


Reaction conditions
 temperature  time  cycle 
 95°C  2min   
 95°C  15sec  25cycle 
 60°C  30sec  25cycle 
 68°C  2min30sec  25cycle 
 68°C  2min30sec   
 14°C  ∞   


PCR product was applied to the 0.7% agarose gel electrophoresis.

Results



WEEK2終わり

August 20th



1. Reproduction of DNA from gel
We did agarose gel electrophoresis (0.7% gel).

Composition
   a product of PCR attB TNFAIP3(8/1) 
 DNA sample  90μL 
 6×Dye  18μL 
 Total  108μL 

We separated 54uL×2 from 108uL (sample) and we did electrophoresis (54uL sample and 1kb marker 5uL).We separated gel containing DNA.

Results
We isolated DNA from these gels by QIA quick Gel Extraction Kit.
Finally we melt DNA to TE Buffer(40uL)

August 21st


TNFA and API2

1. Density measurement of attB TNFAIP3
We did agarose gel electrophoresis (0.7% gel). (1kb marker-3uL, 5times dilution attB TNFAIP3-1uL (make August 20 ), 10times dilution-1uL, 1kb marker-2uL, 15times dilution-1uL, 20times dilution-1uL, 1kb marker-1uL )

Results
We estimated attB TNFAIP3(we make this time) is 35ng/uL



2. BP reaction
We adjusted solution (vials) on 1.5mL tube to next composition.
 attB TNFAIP3(35ng/μL)  2μL 
 pDONR(150ng/μL)  1μL 
 TE buffer  5μL 
 Total  8μL 

We added BP Clonase Ⅱ enzyme mix-2uL to this vials.
We incubated these vials for 2hour at 25˚C. Next we did BP reaction

3. Transformation
We added BP TNFAIP3-2uL to XL1-Blue-100uL and we did Transformation.
Finally we spread 10uL, 40uL and 100uL on plate of LB Kanamycin(+) and we keep 37℃ overnight.

August 22nd


TNFA and API2

We separated six colonies from 40uL spread plate of BP TNFAIP3 (make August 21 ) .
We cultivated these colonies on 2.5mL Kanamycin(+) LB culture medium and we kept shaking this medium overnight.



  • DNA purification and precipitation