Team:Bielefeld-Germany/Labjournal/Molecular

From 2013.igem.org

(Difference between revisions)
Line 23: Line 23:
<tr> <td>Hf-Buffer 5x</td>      <td align="right">10.0</td> <td align="right">88.0</td> </tr>
<tr> <td>Hf-Buffer 5x</td>      <td align="right">10.0</td> <td align="right">88.0</td> </tr>
<tr> <td>dNTPs (10mM)</td>  <td align="right">2.0</td> <td align="right">17.6</td> </tr>
<tr> <td>dNTPs (10mM)</td>  <td align="right">2.0</td> <td align="right">17.6</td> </tr>
-
<tr> <td>Primer1 (10mM)</td> <td align="right">1.0</td> <td align="right">8,8</td> </tr>
+
<tr> <td>Primer1 (10mM)</td> <td align="right">1.0</td> <td align="right">8.8</td> </tr>
-
<tr> <td>Primer2 (10mM)</td> <td align="right">1.0</td> <td align="right">8,8</td> </tr>
+
<tr> <td>Primer2 (10mM)</td> <td align="right">1.0</td> <td align="right">8.8</td> </tr>
<tr> <td>Template (16.5ng)</td> <td align="right">3.0</td> <td align="right">26.4</td> </tr>
<tr> <td>Template (16.5ng)</td> <td align="right">3.0</td> <td align="right">26.4</td> </tr>
<tr> <td>DMSO</td>         <td align="right">4.0</td>    <td align="right">35.2</td> </tr>
<tr> <td>DMSO</td>         <td align="right">4.0</td>    <td align="right">35.2</td> </tr>
Line 63: Line 63:
<tr> <td>Hf-Buffer 5x</td>      <td align="right">10.0</td> <td align="right">88.0</td> </tr>
<tr> <td>Hf-Buffer 5x</td>      <td align="right">10.0</td> <td align="right">88.0</td> </tr>
<tr> <td>dNTPs (10mM)</td>  <td align="right">2.5</td> <td align="right">22.0</td> </tr>
<tr> <td>dNTPs (10mM)</td>  <td align="right">2.5</td> <td align="right">22.0</td> </tr>
-
<tr> <td>Primer1 (10mM)</td> <td align="right">1.0</td> <td align="right">8,8</td> </tr>
+
<tr> <td>Primer1 (10mM)</td> <td align="right">1.0</td> <td align="right">8.8</td> </tr>
-
<tr> <td>Primer2 (10mM)</td> <td align="right">1.0</td> <td align="right">8,8</td> </tr>
+
<tr> <td>Primer2 (10mM)</td> <td align="right">1.0</td> <td align="right">8.8</td> </tr>
<tr> <td>Template (5.5ng/ul)</td><td align="right">2.0</td> <td align="right">17.6</td> </tr>
<tr> <td>Template (5.5ng/ul)</td><td align="right">2.0</td> <td align="right">17.6</td> </tr>
<tr> <td>DMSO</td>         <td align="right">3.0</td>    <td align="right">26.4</td> </tr>
<tr> <td>DMSO</td>         <td align="right">3.0</td>    <td align="right">26.4</td> </tr>
Line 103: Line 103:
<tr> <td>Hf-Buffer 5x</td>      <td align="right">10.0</td> <td align="right">88.0</td> </tr>
<tr> <td>Hf-Buffer 5x</td>      <td align="right">10.0</td> <td align="right">88.0</td> </tr>
<tr> <td>dNTPs (10mM)</td>  <td align="right">2.5</td> <td align="right">22.0</td> </tr>
<tr> <td>dNTPs (10mM)</td>  <td align="right">2.5</td> <td align="right">22.0</td> </tr>
-
<tr> <td>Primer1 (10mM)</td> <td align="right">1.0</td> <td align="right">8,8</td> </tr>
+
<tr> <td>Primer1 (10mM)</td> <td align="right">1.0</td> <td align="right">8.8</td> </tr>
-
<tr> <td>Primer2 (10mM)</td> <td align="right">1.0</td> <td align="right">8,8</td> </tr>
+
<tr> <td>Primer2 (10mM)</td> <td align="right">1.0</td> <td align="right">8.8</td> </tr>
<tr> <td>Template (5.5ng/ul)</td><td align="right">2.0</td> <td align="right">17.6</td> </tr>
<tr> <td>Template (5.5ng/ul)</td><td align="right">2.0</td> <td align="right">17.6</td> </tr>
<tr> <td>DMSO</td>         <td align="right">3.0</td>    <td align="right">26.4</td> </tr>
<tr> <td>DMSO</td>         <td align="right">3.0</td>    <td align="right">26.4</td> </tr>
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       <col width="50">
       <col width="50">
<tr> <th>Reagent</th>         <th>1x</th> <th>8x</th> </tr>
<tr> <th>Reagent</th>         <th>1x</th> <th>8x</th> </tr>
-
<tr> <td>Hf-Buffer 5x</td>      <td align="right">10.0</td> <td align="right">88.0</td> </tr>
+
<tr> <td>GC-Buffer 5x</td>      <td align="right">10.0</td> <td align="right">88.0</td> </tr>
-
<tr> <td>dNTPs (10mM)</td>  <td align="right">2.5</td> <td align="right">22.0</td> </tr>
+
<tr> <td>dNTPs (10mM)</td>  <td align="right">2.0</td> <td align="right">17.6</td> </tr>
-
<tr> <td>Primer1 (10mM)</td> <td align="right">1.0</td> <td align="right">8,8</td> </tr>
+
<tr> <td>Primer1 (10mM)</td> <td align="right">1.25</td> <td align="right">11.0</td> </tr>
-
<tr> <td>Primer2 (10mM)</td> <td align="right">1.0</td> <td align="right">8,8</td> </tr>
+
<tr> <td>Primer2 (10mM)</td> <td align="right">1.25</td> <td align="right">11.0</td> </tr>
-
<tr> <td>Template (5.5ng/ul)</td><td align="right">2.0</td> <td align="right">17.6</td> </tr>
+
<tr> <td>Template (5.5ng/ul)</td><td align="right">1.0</td> <td align="right">8.8</td> </tr>
-
<tr> <td>DMSO</td>         <td align="right">3.0</td>    <td align="right">26.4</td> </tr>
+
<tr> <td>DMSO</td>         <td align="right">1.5</td>    <td align="right">13.2</td> </tr>
-
<tr> <td>Phusion (0.5u)</td>        <td align="right">1.0</td> <td align="right">8.8</td> </tr>
+
<tr> <td>Phusion (0.5u)</td>        <td align="right">0.5</td> <td align="right">4.4</td> </tr>
-
<tr> <td>H2O</td>          <td align="right">29.5</td> <td align="right">259.6</td> </tr>
+
<tr> <td>H2O</td>          <td align="right">32.5</td> <td align="right">286.0</td> </tr>
</table>
</table>
Line 161: Line 161:
<tr><th colspan="5">Program Phusion</th></tr>
<tr><th colspan="5">Program Phusion</th></tr>
<tr align="center"><td>98 °C</td><td>98 °C</td><td>70 °C</td><td>72 °C</td><td>72 °C</td></tr>
<tr align="center"><td>98 °C</td><td>98 °C</td><td>70 °C</td><td>72 °C</td><td>72 °C</td></tr>
-
<tr align="center"><td>30s</td> <td>10s</td><td>30s</td> <td>50s</td><td>6:00</td></tr>
+
<tr align="center"><td>45s</td> <td>30s</td><td>1:00</td> <td>45s</td><td>5:00</td></tr>
<tr><td></td><td colspan="3" align="center">35 cycles</td><td></td></tr>
<tr><td></td><td colspan="3" align="center">35 cycles</td><td></td></tr>
<tr><td colspan="5">
<tr><td colspan="5">

Revision as of 20:32, 30 August 2013




PCR Protocols and Programs

Phusion 50ul for mtrCAB Fragment1

Reagent 1x 8x
Hf-Buffer 5x 10.0 88.0
dNTPs (10mM) 2.0 17.6
Primer1 (10mM) 1.0 8.8
Primer2 (10mM) 1.0 8.8
Template (16.5ng) 3.0 26.4
DMSO 4.0 35.2
Phusion (0.5u) 1.0 8.8
H2O 26.0 228.8
Program Phusion
98 °C98 °C68 °C72 °C72 °C
30s 10s30s 1:356:00
35 cycles
Notes:
Size: 1800bp
Primer1:
Primer2:













Phusion 50ul for mtrCAB Fragment2

Reagent 1x 8x
Hf-Buffer 5x 10.0 88.0
dNTPs (10mM) 2.5 22.0
Primer1 (10mM) 1.0 8.8
Primer2 (10mM) 1.0 8.8
Template (5.5ng/ul)2.0 17.6
DMSO 3.0 26.4
Phusion (0.5u) 1.0 8.8
H2O 29.5 259.6
Program Phusion
98 °C98 °C60 °C72 °C72 °C
1:00 10s30s 30s6:00
35 cycles
Notes:
Size: 330bp
Primer1:
Primer2:













Phusion 50ul for mtrCAB Fragment3

Reagent 1x 8x
Hf-Buffer 5x 10.0 88.0
dNTPs (10mM) 2.5 22.0
Primer1 (10mM) 1.0 8.8
Primer2 (10mM) 1.0 8.8
Template (5.5ng/ul)2.0 17.6
DMSO 3.0 26.4
Phusion (0.5u) 1.0 8.8
H2O 29.5 259.6
Program Phusion
98 °C98 °C70 °C72 °C72 °C
30s 10s30s 50s6:00
35 cycles
Notes:
Size: 3000bp
Primer1:
Primer2:













Phusion 50ul for psB1C3 with Gibson-overlap for mtrCAB

Reagent 1x 8x
GC-Buffer 5x 10.0 88.0
dNTPs (10mM) 2.0 17.6
Primer1 (10mM) 1.25 11.0
Primer2 (10mM) 1.25 11.0
Template (5.5ng/ul)1.0 8.8
DMSO 1.5 13.2
Phusion (0.5u) 0.5 4.4
H2O 32.5 286.0
Program Phusion
98 °C98 °C70 °C72 °C72 °C
45s 30s1:00 45s5:00
35 cycles
Notes:
Size: 2070bp
Primer1:
Primer2:













Taq 25 µl

Reagenz 1x 8x
Buffer 5x 5,0 44
MgSO4 0 0
dNTPs 2,5 22
Primer1 1,0 8,8
Primer2 1,0 8,8
Template 1,5 13,2
DMSO 2,0 17,6
Polymerase0,5 4,4
H2O 11,5 101,2
Program Taq
98 °C98 °Cx °C72 °C72 °C
2 min 30 s30 s y6 min
x: according to Primer Tm
y: 15s/kb

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