"

From 2013.igem.org

Labbook |	Protocols |	Materials |

Contents 1 The detection construct pSB1C3-pBAD-CheB-mKate-terminator-pBAD-tar-LssmOrange 1.1 'traditional cloning approach' 1.2 'gBLOCK approach' 1.3 'InFusion approach'

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