Team:TU Darmstadt/labbook/DetectionConstruct

From 2013.igem.org

(Difference between revisions)
Line 53: Line 53:
!align="center"|[[Team:TU_Darmstadt/materials|<span style="color:white;font-size:160%;"> Materials |</span>]]
!align="center"|[[Team:TU_Darmstadt/materials|<span style="color:white;font-size:160%;"> Materials |</span>]]
|}
|}
 +
 +
==='''The detection construct pSB1C3-pBAD-CheB-mKate-terminator-pBAD-tar-LssmOrange'''===
 +
 +
====''''traditional cloning approach''''====
 +
was abbanndoned due to major difficulties during restriction, ligation and transformation
 +
 +
====''''gBLOCK approach''''====
 +
was abbanndoned due to major difficulties during restriction, ligation and transformation
 +
 +
====''''InFusion approach''''====
 +
<html>
 +
<head>
 +
<title></title>
 +
<meta name="author" content="Anne Schieferdecker">
 +
<meta name="editor" content="html-editor phase 5">
 +
</head>
 +
<body>
 +
      <table border="1" rules="rows"  >
 +
        <tr>
 +
                <td>13.08</td>
 +
                <td>gBLOCK assembly of CMK and TLO</td>
 +
                <td></td>
 +
        </tr>
 +
        <tr>
 +
                <td></td>
 +
                <td><ul><li>PCR mixture
 +
                                <ul><li>1 µL of each gBLOCK
 +
                                <ul>
 +
                                        <li>CMK: B_01 - B_04</li>
 +
                                        <li>TLO: A_01 - A_06</li>
 +
                                </ul></li>
 +
                                <li>10 µL 5x Q5 Reaction Buffer</li>
 +
                                <li>2 µL dNTPs</li>
 +
                                <li>1 µL Q5 Hot Start Polymerase</li>
 +
                                <li>10 µL 5x Q5 High GC Enhancer</li>
 +
                                <li>1 µL primer suffix-R (10 mM)</li>
 +
                                <li>1 µL primer prefix_R (10 mM)</li>
 +
                        </ul></ul></li>
 +
                        <ul><li>PCR program (40 cycles)
 +
                                <ul><li>initial denaturation 94°C, 100s</li>
 +
                                <li>denaturation 94°C, 55s</li>
 +
                                <li>annealing 64°C, 55s</li>
 +
                                <li>elongation 72°C, 120s</li>
 +
                                <li>final elongation 72°C, 300s</li>
 +
                        </li></ul></ul>
 +
                        <ul><li>preparative 1% agarose gel
 +
                                <ul><li>gel displays bands of expected size, therefore assembly was successful</li>
 +
                        </ul></li></ul>
 +
                </td>
 +
                <td><img src="https://static.igem.org/mediawiki/2013/6/6c/Darmstadt13_InFusion_130813.png" alt=""</td>
 +
        </tr>
 +
      </table>
 +
</body>
 +
</html>

Revision as of 11:53, 3 October 2013

Labbook | Protocols | Materials |

Contents

The detection construct pSB1C3-pBAD-CheB-mKate-terminator-pBAD-tar-LssmOrange

'traditional cloning approach'

was abbanndoned due to major difficulties during restriction, ligation and transformation

'gBLOCK approach'

was abbanndoned due to major difficulties during restriction, ligation and transformation

'InFusion approach'

13.08 gBLOCK assembly of CMK and TLO
  • PCR mixture
    • 1 µL of each gBLOCK
      • CMK: B_01 - B_04
      • TLO: A_01 - A_06
    • 10 µL 5x Q5 Reaction Buffer
    • 2 µL dNTPs
    • 1 µL Q5 Hot Start Polymerase
    • 10 µL 5x Q5 High GC Enhancer
    • 1 µL primer suffix-R (10 mM)
    • 1 µL primer prefix_R (10 mM)
  • PCR program (40 cycles)
    • initial denaturation 94°C, 100s
    • denaturation 94°C, 55s
    • annealing 64°C, 55s
    • elongation 72°C, 120s
    • final elongation 72°C, 300s
  • preparative 1% agarose gel
    • gel displays bands of expected size, therefore assembly was successful

Retrieved from "http://2013.igem.org/Team:TU_Darmstadt/labbook/DetectionConstruct"