Team:Heidelberg/Templates/DelH week24

From 2013.igem.org

(Difference between revisions)
(Created page with " == 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''' {| cla...")
 
(3 intermediate revisions not shown)
Line 6: Line 6:
{| class="wikitable"
{| class="wikitable"
|-
|-
-
! Reagent !! Amount   
+
! Reagent !! Amount [µl]    
|-
|-
-
| ''D. acidovorans'' SPH-1 colony || 1
+
| pSB6A1 || 1
|-
|-
-
| SR_01:  (1/10) || 2
+
| FS_85:  (1/10) || 1
|-
|-
-
| FS_23:  (1/10) || 2
+
| FS_94:  (1/10) || 1
|-
|-
| Phusion flash Master Mix || 10
| Phusion flash Master Mix || 10
Line 31: Line 31:
| 1 || 98 || 10
| 1 || 98 || 10
|-
|-
-
| rowspan="3"| 12 || 98 || 1
+
| rowspan="2"| 35 || 98 || 1
|-
|-
-
| 66 ↓ 0.5 || 5
+
| 72 || 1:25
|-
|-
-
| 72 || 3:20
+
| 1 || 72 || 10min
|-
|-
-
| rowspan="2"| 18 || 98 || 1
+
| 1  || 12 || inf
 +
|}
 +
 
 +
'''Results:'''
 +
 
 +
====Amplification of pFS_02 Backbone (HM_17 to FS_78; 4.1 kbp)====
 +
:'''Reaction'''
 +
{| class="wikitable"
 +
|-
 +
! Reagent !! Amount [µl] 
 +
|-
 +
| pSB6A1 || 1
 +
|-
 +
| HM_17:  (1/10) || 1
 +
|-
 +
| FS_78:  (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
 +
|}
 +
 
 +
'''Results:'''
 +
 
 +
====Amplification of pFS_02 Backbone (HM_17 to FS_85; 4.1 kbp)====
 +
:'''Reaction'''
 +
{| class="wikitable"
 +
|-
 +
! Reagent !! Amount [µl] 
 +
|-
 +
| pSB6A1 || 1
 +
|-
 +
| HM_17:  (1/10) || 1
 +
|-
 +
| FS_85:  (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
 +
|}
 +
 
 +
'''Results:'''
 +
 
 +
====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="2"| 35 || 98 || 1
 +
|-
 +
| 72  || 1:25
 +
|-
 +
| 1 || 72 || 10min
 +
|-
 +
| 1  || 12 || inf
 +
|}
 +
 
 +
'''Results:'''
 +
 
 +
====Amplification of DelH fragment (FS_84 to FS_66; 4.1 kbp)====
 +
:'''Reaction'''
 +
{| class="wikitable"
 +
|-
 +
! Reagent !! Amount [µl] 
 +
|-
 +
| ''D. acidovorans'' SPH-1 || 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="2"| 35 || 98 || 1
 +
|-
 +
| 72  || 1:25
 +
|-
 +
| 1 || 72 || 10min
 +
|-
 +
| 1  || 12 || inf
 +
|}
 +
 
 +
'''Results:'''
 +
 
 +
====Amplification of ccdB fragment for pFS_04 (FS_90 to FS_93; 700 bp)====
 +
:'''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
|-
|-
-
| 64  || 5
+
| rowspan="2"| 35 || 98 || 1
|-
|-
-
| 72  || 3:20
+
| 72  || 1:25
|-
|-
| 1 || 72 || 10min
| 1 || 72 || 10min

Latest revision as of 15:35, 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
35 98 1
72 1:25
1 72 10min
1 12 inf

Results:

Amplification of pFS_02 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
72 1:25
1 72 10min
1 12 inf

Results:

Amplification of pFS_02 Backbone (HM_17 to FS_85; 4.1 kbp)

Reaction
Reagent Amount [µl]
pSB6A1 1
HM_17: (1/10) 1
FS_85: (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

Results:

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
72 1:25
1 72 10min
1 12 inf

Results:

Amplification of DelH fragment (FS_84 to FS_66; 4.1 kbp)

Reaction
Reagent Amount [µl]
D. acidovorans SPH-1 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
72 1:25
1 72 10min
1 12 inf

Results:

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

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

Results:

Retrieved from "http://2013.igem.org/Team:Heidelberg/Templates/DelH_week24"