Team:Heidelberg/Templates/DelH week25

From 2013.igem.org

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== 23-10 - 29-10-13 ==
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 +
== 14-10 - 20-10-13 ==
===Generation of DelH plasmids pFS02, pFS03, pFS04 and pFS05===
===Generation of DelH plasmids pFS02, pFS03, pFS04 and pFS05===
====Amplification of pFS_04 Backbone (FS_85 to FS_94; 4.1 kbp)====
====Amplification of pFS_04 Backbone (FS_85 to FS_94; 4.1 kbp)====
Line 93: Line 94:
| 1  || 12 || inf
| 1  || 12 || inf
|}
|}
 +
 +
'''Results:'''
 +
 +
* Amplification of Backbone for pFS_03 worked
 +
* repeat PCR to obtain amount of DNA required for Gibson Assembly
====Amplification of pFS_04 Backbone (FS_85 to FS_94; 4.1 kbp)====
====Amplification of pFS_04 Backbone (FS_85 to FS_94; 4.1 kbp)====
Line 134: Line 140:
====Amplification of pFS_03 Backbone (FS_86 to FS_87; 4.1 kbp)====
====Amplification of pFS_03 Backbone (FS_86 to FS_87; 4.1 kbp)====
 +
4 reactions of 20µl each carried out
:'''Reaction'''  
:'''Reaction'''  
{| class="wikitable"
{| class="wikitable"
Line 173: Line 180:
|}
|}
-
====Amplification of pFS_03 Backbone (HM_17 to FS_78; 4.1 kbp)====
+
'''Results:'''  
-
:'''Reaction'''  
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-
{| class="wikitable"
+
-
|-
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-
! Reagent !! Amount [µl] 
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-
|-
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-
| pSB6A1 || 1
+
-
|-
+
-
| HM_17:  (1/10) || 1
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-
|-
+
-
| FS_78:  (1/10) || 1
+
-
|-
+
-
| Phusion flash Master Mix || 10
+
-
|-
+
-
| DMSO || 1
+
-
|-
+
-
| dd H<sub>2</sub>O || 4
+
-
|}
+
-
:'''Conditions '''
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* Amplification of Backbone for pFS_03 worked
-
 
+
* Gibson Assembly will be conducted
-
{| class="wikitable"
+
-
|-
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-
! colspan="3" | Biorad T100
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-
|-
+
-
! Cycles !! temperature [°C] !! Time [s]
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-
|-
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-
| 1 || 98 || 10
+
-
|-
+
-
| rowspan="3"| 35 || 98 || 1
+
-
|-
+
-
| 58  || 5
+
-
|-
+
-
| 72  || 1:25
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-
|-
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-
| 1 || 72 || 10min
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-
|-
+
-
| 1  || 12 || inf
+
-
|}
+
====Amplification of ccdB fragment for pFS_04 (FS_90 to FS_93; 700 bp)====
====Amplification of ccdB fragment for pFS_04 (FS_90 to FS_93; 700 bp)====
Line 394: Line 366:
-
====Amplification of Backbone fragment for pFS_02 (HM_17 to FS_87; 4.2 kbp)====
+
 
-
4 reactions with 20µl each, followed by DpnI digest
+
 
-
:'''Reaction'''
+
==Gibson assembly of pFS_03==
-
{| class="wikitable"
+
{|class="wikitable"
|-
|-
-
! Reagent !! Amount [µl] 
+
! Reagent !! Volume added (µl)
|-
|-
-
| pSB6A1 || 0.2
+
| BB (FS_86 - FS_87) || 0.61
|-
|-
-
| HM_17:  (1/10) || 1
+
| DN11-FS66 || 1.00
|-
|-
-
| FS_87:  (1/10) || 1
+
| FS_67-FS_68 || 1.35
|-
|-
-
| Phusion flash Master Mix || 10
+
| FS_69-FS_70 || 6.34
|-
|-
-
| DMSO || 1
+
| FS_71-HM08 || 0.70
|-
|-
-
| dd H<sub>2</sub>O || 4.8
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| Gibson Master Mix 2x || 10
|}
|}
-
:'''Conditions'''
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====Colony-PCR, screening for pFS_03====
-
 
+
{| class="wikitable" style="float:left; margin-right:1em"
-
{| class="wikitable"
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|-
|-
-
! colspan="3" | Biorad T100
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| Template || 60x1 picked colony
|-
|-
-
! Cycles !! temperature [°C] !! Time [s]
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| Expected length [bp] ||  521
|-
|-
-
| 1 || 98 || 10
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| Named || 1A - 12H, DelH_pSB6A1_weak_promotor
|-
|-
-
| rowspan="3"| 12 || 98 || 1
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| Primer fw 10 µM ||60x 0.5 µl FS_73 
|-
|-
-
| 68 ↓ 0.5 || 5
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| Primer rev 10 µM || 60x 0.5 µl FS_95
|-
|-
-
| 72  || 1:25
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| One-Taq Polymerase (2x) ||60x 5 µl
|-
|-
-
| rowspan="3"| 18 || 98 || 1
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| ddH<sub>2</sub>O || 60x 4 µl 
 +
|}
 +
{| class="wikitable" style="float:left; margin-right:1em"
|-
|-
-
| 66  || 5
+
! Cycles !! Temperature [°C] !! Time [s]
|-
|-
-
| 72  || 1:25
+
| 1 || 95 || 120
|-
|-
-
| 1 || 72 || 10min
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| rowspan="3" | 12 || 95 || 30
|-
|-
-
| 1  || 12 || inf
+
| 68 (touchdown -0.5°C) || 30
 +
|-
 +
| 72 || 45 s
 +
|-
 +
| rowspan="3" | 18 ||  95 || 30
 +
|-
 +
| 65  || 30
 +
|-
 +
| 72 || 45
 +
|-
 +
| 1  || 12 || inf  
|}
|}
 +
<div style="clear:both"></div>
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==Gibson assembly of pFS_04==
-
=18.10=
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-
==HM17-FS_78==
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Annealing temps: 62°C, 60°C, 57°C, 55°C <br/>
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Elongation time: 2:10 <br/>
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--> Did not work
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==Screening remaining Gibson electroporated==
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60 colonies were picked and screened with primers FS_73 and FS_95 (annealing 66 touchdown, 62)
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-
==Gibson assembly==
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{|class="wikitable"
{|class="wikitable"
|-
|-
! Reagent !! Volume added (µl)
! Reagent !! Volume added (µl)
|-
|-
-
| BB (FS..) || 0.61
+
| BB (FS_85-FS_94) || 0.44
|-
|-
-
| DN11-FS66 || 1.00
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| ccdB (FS_90-FS_93 || 0.15
|-
|-
-
| FS_67-FS_68 || 1.35
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| ddH<sub>2</sub>O || 9.41
-
|-
+
-
| FS_69-FS_70 || 6.34
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-
|-
+
-
| FS_71-HM08 || 0.70
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|-
|-
| Gibson Master Mix 2x || 10
| Gibson Master Mix 2x || 10
|}
|}
-
==ccdB==
 
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Annealing temps: </br>
 
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12 cycles 74, 68, 64 and 58°C <br/>
 
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18 cycles 72, 66, 62 and 56°C
 
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=20-10-2013=
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====Colony-PCR, screening for pFS_04====
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==Inocultation triple dummy clone==
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{| class="wikitable" style="float:left; margin-right:1em"
-
==Screening triple clone==
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|-
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each with positive control
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| Template || 30x1 picked colony
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IK25+VF2 -->54°C, 90s elongation <br/>
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|-
-
FS47+FS48 --> 66t,62 40s elongation <br/>
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| Expected length [bp] ||  ~800bp
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FS95+FS73 --> 66t,62 40s elongation <br/>
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|-
 +
| Named || 1A - 6C, pSB6A1_ccdB
 +
|-
 +
| Primer fw 10 µM ||30x 0.5 µl KH_9 
 +
|-
 +
| Primer rev 10 µM || 30x 0.5 µl VF2  
 +
|-
 +
| One-Taq Polymerase (2x) ||30x 5 µl
 +
|-
 +
| ddH<sub>2</sub>O || 30x 4 µl 
 +
|}
 +
{| class="wikitable" style="float:left; margin-right:1em"
 +
|-
 +
! Cycles !! Temperature [°C] !! Time [s]
 +
|-
 +
| 1 || 95 || 120
 +
|-
 +
| rowspan="3" | 35 ||  95 || 30
 +
|-
 +
| 57  || 30
 +
|-
 +
| 72 || 45
 +
|-
 +
| 1  || 12 || inf
 +
|}
 +
<div style="clear:both"></div>
 +
The colony PCR was positive for several clones. Those clones were cultivated and preped.
 +
====Restriction Digest with PvuI====
 +
The restriction digest was positive for colonies B9 and B10.
 +
====Sequencing of clones B9 and B10 with DN07====
 +
The sequencing was positive for clones B9 and B10.
 +
<gallery>
 +
File:B9 DN07.txt| Sequencing clone B9 with DN07
 +
File:B10 DN07.txt| Sequencing clone B10 with DN07
 +
File:Alignement B9 DN07.txt | Alignement of sequencing of clone B9 with DN07
 +
File:Alignement B10 DN07.txt | Alignement of sequencing of clone B10 with DN07
 +
</gallery>

Latest revision as of 03:43, 29 October 2013

Contents

14-10 - 20-10-13

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 1:25
18 98 1
66 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
12 98 1
68 ↓ 0.5 5
72 1:25
18 98 1
66 5
72 1:25
1 72 10min
1 12 inf

Results:

  • Amplification of Backbone for pFS_03 worked
  • repeat PCR to obtain amount of DNA required for Gibson Assembly

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)

4 reactions of 20µl each carried out

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

Results:

  • Amplification of Backbone for pFS_03 worked
  • Gibson Assembly will be conducted

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 0:18
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 1:25
18 98 1
66 5
72 1:25
1 72 10min
1 12 inf

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

4 reactions with 20µl each, followed by DpnI digest

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 1:25
18 98 1
66 5
72 1:25
1 72 10min
1 12 inf

Amplification of DelH fragment (FS_69 to FS_70; 7.0 kbp)

7 reactions with 20µl each

Reaction
Reagent Amount [µl]
D. acidovorans SPH-1 1
FS_69: (1/10) 1
FS_70: (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 1:25
18 98 1
66 5
72 1:25
1 72 10min
1 12 inf



Gibson assembly of pFS_03

Reagent Volume added (µl)
BB (FS_86 - FS_87) 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

Colony-PCR, screening for pFS_03

Template 60x1 picked colony
Expected length [bp] 521
Named 1A - 12H, DelH_pSB6A1_weak_promotor
Primer fw 10 µM 60x 0.5 µl FS_73
Primer rev 10 µM 60x 0.5 µl FS_95
One-Taq Polymerase (2x) 60x 5 µl
ddH2O 60x 4 µl
Cycles Temperature [°C] Time [s]
1 95 120
12 95 30
68 (touchdown -0.5°C) 30
72 45 s
18 95 30
65 30
72 45
1 12 inf

Gibson assembly of pFS_04

Reagent Volume added (µl)
BB (FS_85-FS_94) 0.44
ccdB (FS_90-FS_93 0.15
ddH2O 9.41
Gibson Master Mix 2x 10


Colony-PCR, screening for pFS_04

Template 30x1 picked colony
Expected length [bp] ~800bp
Named 1A - 6C, pSB6A1_ccdB
Primer fw 10 µM 30x 0.5 µl KH_9
Primer rev 10 µM 30x 0.5 µl VF2
One-Taq Polymerase (2x) 30x 5 µl
ddH2O 30x 4 µl
Cycles Temperature [°C] Time [s]
1 95 120
35 95 30
57 30
72 45
1 12 inf

The colony PCR was positive for several clones. Those clones were cultivated and preped.

Restriction Digest with PvuI

The restriction digest was positive for colonies B9 and B10.

Sequencing of clones B9 and B10 with DN07

The sequencing was positive for clones B9 and B10.

Sequencing clone B9 with DN07

Sequencing clone B10 with DN07

Alignement of sequencing of clone B9 with DN07

Alignement of sequencing of clone B10 with DN07

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