Team:Heidelberg/Templates/DelH week25

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Contents

23-09 - 29-09-13

Generation of DelH plasmids pFS02, pFS03, pFS04 and pFS05

Amplification of pFS_04 Backbone (FS_85 to FS_94; 4.1 kbp)

Reaction
Reagent Amount [µl]
pSB6A1 1
FS_85: (1/10) 1
FS_94: (1/10) 1
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 3:20
18 98 1
66 5
72 3:20
1 72 10min
1 12 inf


Amplification of pFS_03 Backbone (FS_85 to FS_94; 4.1 kbp)

Reaction
Reagent Amount [µl]
pSB4K5 1
FS_85: (1/10) 1
FS_94: (1/10) 1
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 3:20
18 98 1
66 5
72 3:20
1 72 10min
1 12 inf

Amplification of pFS_04 Backbone (FS_85 to FS_94; 4.1 kbp)

Reaction
Reagent Amount [µl]
pSB6A1 1
FS_85: (1/10) 1
FS_94: (1/10) 1
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
35 98 1
58 5
72 1:25
1 72 10min
1 12 inf


18.10

HM17-FS_78

Annealing temps: 62°C, 60°C, 57°C, 55°C
Elongation time: 2:10
--> Did not work

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
18 cycles 72, 66, 62 and 56°C

20-10-2013

Inocultation triple dummy clone

Screening triple clone

each with positive control IK25+VF2 -->54°C, 90s elongation
FS47+FS48 --> 66t,62 40s elongation
FS95+FS73 --> 66t,62 40s elongation