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From 2013.igem.org

(18.10)

Contents 1 HM17-FS_78 2 Screening remaining Gibson electroporated 3 Gibson assembly 4 ccdB

HM17-FS_78

Annealing temps: 62°C, 60°C, 57°C, 55°C
Elongation time: 2:10
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Screening remaining Gibson electroporated

60 colonies were picked and screened with primers FS_73 and FS_95 (annealing 66 touchdown, 62)

Gibson assembly

Reagent	Volume added (µl)
BB (FS..)	0.61
DN11-FS66	1.00
FS_67-FS_68	1.35
FS_69-FS_70	6.34
FS_71-HM08	0.70
Gibson Master Mix 2x	10

ccdB

Annealing temps: </br> 12 cycles 74, 68, 64 and 58°C </br> 18 cycles 72, 66, 62 and 56°C