Team:Heidelberg/Templates/DelH week25

From 2013.igem.org

(Difference between revisions)
Line 212: Line 212:
| 1  || 12 || inf
| 1  || 12 || inf
|}
|}
 +
 +
====Amplification of ccdB fragment for pFS_04 (FS_90 to FS_93; 700 bp)====
 +
4 reactions with 20µl each, followed by DpnI digest
 +
:'''Reaction'''
 +
{| class="wikitable"
 +
|-
 +
! Reagent !! Amount [µl] 
 +
|-
 +
| pDonor-Plasmid || 1
 +
|-
 +
| FS_90:  (1/10) || 1
 +
|-
 +
| FS_93:  (1/10) || 1
 +
|-
 +
| Phusion flash Master Mix || 10
 +
|-
 +
| DMSO || 1
 +
|-
 +
| dd H<sub>2</sub>O || 4
 +
|}
 +
 +
:'''Conditions '''
 +
 +
{| class="wikitable"
 +
|-
 +
! colspan="3" | Biorad T100
 +
|-
 +
! Cycles !! temperature [°C] !! Time [s]
 +
|-
 +
| 1 || 98 || 10
 +
|-
 +
| rowspan="2"| 35 || 98 || 1
 +
|-
 +
| 72  || 1:25
 +
|-
 +
| 1 || 72 || 10min
 +
|-
 +
| 1  || 12 || inf
 +
|}
 +
 +
====Amplification of pFS_04 Backbone (FS_85 to FS_94; 4.1 kbp)====
 +
4 reactions with 20µl each, followed by DpnI digest
 +
:'''Reaction'''
 +
{| class="wikitable"
 +
|-
 +
! Reagent !! Amount [µl] 
 +
|-
 +
| pSB6A1 || 1
 +
|-
 +
| FS_85:  (1/10) || 1
 +
|-
 +
| FS_94:  (1/10) || 1
 +
|-
 +
| Phusion flash Master Mix || 10
 +
|-
 +
| DMSO || 1
 +
|-
 +
| dd H<sub>2</sub>O || 4
 +
|}
 +
 +
:'''Conditions'''
 +
 +
{| class="wikitable"
 +
|-
 +
! colspan="3" | Biorad T100
 +
|-
 +
! Cycles !! temperature [°C] !! Time [s]
 +
|-
 +
| 1 || 98 || 10
 +
|-
 +
| rowspan="3"| 12 || 98 || 1
 +
|-
 +
| 68 ↓ 0.5  || 5
 +
|-
 +
| 72  || 3:20
 +
|-
 +
| rowspan="3"| 18 || 98 || 1
 +
|-
 +
| 66  || 5
 +
|-
 +
| 72  || 3:20
 +
|-
 +
| 1 || 72 || 10min
 +
|-
 +
| 1  || 12 || inf
 +
|}
 +
 +
====Amplification of pFS_03 Backbone (FS_86 to FS_87; 4.1 kbp)====
 +
:'''Reaction'''
 +
{| class="wikitable"
 +
|-
 +
! Reagent !! Amount [µl] 
 +
|-
 +
| pSB4K5 || 1
 +
|-
 +
| FS_86:  (1/10) || 1
 +
|-
 +
| FS_87:  (1/10) || 1
 +
|-
 +
| Phusion flash Master Mix || 10
 +
|-
 +
| DMSO || 1
 +
|-
 +
| dd H<sub>2</sub>O || 4
 +
|}
 +
 +
:'''Conditions'''
 +
 +
{| class="wikitable"
 +
|-
 +
! colspan="3" | Biorad T100
 +
|-
 +
! Cycles !! temperature [°C] !! Time [s]
 +
|-
 +
| 1 || 98 || 10
 +
|-
 +
| rowspan="3"| 12 || 98 || 1
 +
|-
 +
| 68 ↓ 0.5  || 5
 +
|-
 +
| 72  || 3:20
 +
|-
 +
| rowspan="3"| 18 || 98 || 1
 +
|-
 +
| 66  || 5
 +
|-
 +
| 72  || 3:20
 +
|-
 +
| 1 || 72 || 10min
 +
|-
 +
| 1  || 12 || inf
 +
|}
 +
 +
 +
=18.10=
=18.10=

Revision as of 15:46, 22 October 2013

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_86 to FS_87; 4.1 kbp)

Reaction
Reagent Amount [µl]
pSB4K5 1
FS_86: (1/10) 1
FS_87: (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

Amplification of pFS_03 Backbone (FS_86 to FS_87; 4.1 kbp)

Reaction
Reagent Amount [µl]
pSB4K5 1
FS_86: (1/10) 1
FS_87: (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

Amplification of pFS_03 Backbone (HM_17 to FS_78; 4.1 kbp)

Reaction
Reagent Amount [µl]
pSB6A1 1
HM_17: (1/10) 1
FS_78: (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

Amplification of ccdB fragment for pFS_04 (FS_90 to FS_93; 700 bp)

4 reactions with 20µl each, followed by DpnI digest

Reaction
Reagent Amount [µl]
pDonor-Plasmid 1
FS_90: (1/10) 1
FS_93: (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
72 1:25
1 72 10min
1 12 inf

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

4 reactions with 20µl each, followed by DpnI digest

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_86 to FS_87; 4.1 kbp)

Reaction
Reagent Amount [µl]
pSB4K5 1
FS_86: (1/10) 1
FS_87: (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



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

Retrieved from "http://2013.igem.org/Team:Heidelberg/Templates/DelH_week25"