Team:Uppsala-SwedenWeek16

From 2010.igem.org

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The purpose of  this was to get more reliable band when doing a PCR and running the gel.
The purpose of  this was to get more reliable band when doing a PCR and running the gel.
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<TABLE WIDTH=263 BORDER=1 BORDERCOLOR="#000000" CELLPADDING=7 CELLSPACING=0>
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<COL WIDTH=134>
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<COL WIDTH=99>
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<TR VALIGN=TOP>
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<TD WIDTH=134 HEIGHT=8>
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<P LANG="sv-SE" CLASS="western">Mastermix</P>
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</TD>
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<TD WIDTH=99>
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<P CLASS="western"><SPAN LANG="sv-SE">Volume (µL)</SPAN></P>
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</TD>
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</TR>
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<TR VALIGN=TOP>
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<TD WIDTH=134>
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<P LANG="sv-SE" CLASS="western">H20</P>
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</TD>
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<TD WIDTH=99>
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<P LANG="sv-SE" CLASS="western">10.8</P>
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</TD>
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</TR>
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<TR VALIGN=TOP>
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<TD WIDTH=134>
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<P CLASS="western"><SPAN LANG="sv-SE">5X</SPAN><SPAN LANG="sv-SE">
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phusion HF Buffer</SPAN></P>
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</TD>
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<TD WIDTH=99>
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<P LANG="sv-SE" CLASS="western">4</P>
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</TD>
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</TR>
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<TR VALIGN=TOP>
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<TD WIDTH=134>
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<P CLASS="western"><SPAN LANG="sv-SE">d</SPAN><SPAN LANG="sv-SE">NTPs</SPAN></P>
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</TD>
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<TD WIDTH=99>
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<P LANG="sv-SE" CLASS="western">2</P>
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</TD>
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</TR>
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<TR VALIGN=TOP>
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<TD WIDTH=134>
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<P LANG="sv-SE" CLASS="western">VF2</P>
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</TD>
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<TD WIDTH=99>
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<P LANG="sv-SE" CLASS="western">1</P>
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</TD>
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</TR>
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<TR VALIGN=TOP>
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<TD WIDTH=134>
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<P LANG="sv-SE" CLASS="western">VR</P>
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</TD>
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<TD WIDTH=99>
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<P LANG="sv-SE" CLASS="western">1</P>
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</TD>
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</TR>
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<TR VALIGN=TOP>
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<TD WIDTH=134>
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<P CLASS="western"><SPAN LANG="sv-SE">DN</SPAN><SPAN LANG="sv-SE">A-template</SPAN></P>
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</TD>
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<TD WIDTH=99>
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<P LANG="sv-SE" CLASS="western">1</P>
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</TD>
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</TR>
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<TR VALIGN=TOP>
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<TD WIDTH=134>
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<P LANG="sv-SE" CLASS="western">Phusion Polymerase</P>
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</TD>
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<TD WIDTH=99>
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<P LANG="sv-SE" CLASS="western">0.2</P>
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</TD>
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</TR>
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</TABLE>

Revision as of 19:41, 26 October 2010

Week-16

Start to apply high fidelity PCR due the construct are getting larger and the regular Taq polymerase may not perform as well as the Phusion Polymerase at this length. Based on the company´s manual, we calculated the


Temperatur

Time

Cycle

Initial denaturation

98

30 s

1

Denaturation

98

10

30

Annealing

55.3

10s

30

Extension

72

1 min 45 s

30

Final extension

72

5 min

1


4

forever



The purpose of this was to get more reliable band when doing a PCR and running the gel.

<COL WIDTH=134> <COL WIDTH=99>

Mastermix

Volume (µL)

H20

10.8

5X phusion HF Buffer

4

dNTPs

2

VF2

1

VR

1

DNA-template

1

Phusion Polymerase

0.2
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