Team:TU Darmstadt/labbook/DetectionConstruct

From 2013.igem.org

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==='''The detection construct pSB1C3-pBAD-CheB-mKate-terminator-pBAD-tar-LssmOrange'''===
==='''The detection construct pSB1C3-pBAD-CheB-mKate-terminator-pBAD-tar-LssmOrange'''===
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====''''traditional cloning approach''''====
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===='''traditional cloning approach'''====
was abbanndoned due to major difficulties during restriction, ligation and transformation
was abbanndoned due to major difficulties during restriction, ligation and transformation
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====''''gBLOCK approach''''====
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===='''gBLOCK approach'''====
was abbanndoned due to major difficulties during restriction, ligation and transformation
was abbanndoned due to major difficulties during restriction, ligation and transformation
-
====''''InFusion approach''''====
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===='''InFusion approach'''====
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Revision as of 11:55, 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

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