Team:Heidelberg/Templates/Del week10 AF

From 2013.igem.org

(Difference between revisions)
(Created page with "==03-07-2013== ===Amplification of FS_02 to FS_05; 11.2 kb=== [[File:20130703 DelAF DelFG.png|150px|thumb|Amplification of DelA-F and DelF-G, 03-07-13; run at 130 V, 0.8 % gel (T...")
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==03-07-2013==
==03-07-2013==
-
===Amplification of FS_02 to FS_05; 11.2 kb===
+
 
 +
===Amplification from FS_02 to FS_05; 11.2 kb===
 +
 
[[File:20130703 DelAF DelFG.png|150px|thumb|Amplification of DelA-F and DelF-G, 03-07-13; run at 130 V, 0.8 % gel (TAE)]]
[[File:20130703 DelAF DelFG.png|150px|thumb|Amplification of DelA-F and DelF-G, 03-07-13; run at 130 V, 0.8 % gel (TAE)]]
 +
[[File:20130703 DelAF DelFG cut.png|150px|thumb|Gel after removing the DelA-F-fragment, 03-07-13.]]
[[File:20130703 DelAF DelFG cut.png|150px|thumb|Gel after removing the DelA-F-fragment, 03-07-13.]]
 +
:'''Reaction'''
:'''Reaction'''
 +
{| class="wikitable"
{| class="wikitable"
 +
|-
|-
 +
!  µL 1st PCR !! what !!  µL 2nd PCR  
!  µL 1st PCR !! what !!  µL 2nd PCR  
 +
|-
|-
 +
| 1 ||  ''D. acidovorans'' || 1
| 1 ||  ''D. acidovorans'' || 1
 +
|-
|-
 +
| 2.5 || FS_02 (1/10) || 2.5
| 2.5 || FS_02 (1/10) || 2.5
 +
|-
|-
 +
| 2.5 || FS_05 (1/10) || 2.5
| 2.5 || FS_05 (1/10) || 2.5
 +
|-
|-
 +
| 25  || Phusion Master Mix || 25
| 25  || Phusion Master Mix || 25
 +
|-
|-
 +
| - || DMSO || 2.5
| - || DMSO || 2.5
 +
|-
|-
 +
| 19  || dd H<sub>2</sub>O || 19
| 19  || dd H<sub>2</sub>O || 19
 +
|}
|}
 +
 +
:'''Conditions'''
:'''Conditions'''
 +
{| class="wikitable"
{| class="wikitable"
 +
|-
|-
 +
! colspan="3" | Biorad MyCycler
! colspan="3" | Biorad MyCycler
 +
|-
|-
 +
! Cycles-PCR !!  temperature [°C] !! Time [s]
! Cycles-PCR !!  temperature [°C] !! Time [s]
 +
|-
|-
 +
| 1 || 98 || 10
| 1 || 98 || 10
 +
|-
|-
 +
| rowspan="3"| 12 || 98 || 1
| rowspan="3"| 12 || 98 || 1
 +
|-
|-
 +
| 68 ↓ 0.5 || 5
| 68 ↓ 0.5 || 5
 +
|-
|-
 +
| 72 || 3 min
| 72 || 3 min
 +
|-
|-
 +
|rowspan="3"| 18 || 98 || 1
|rowspan="3"| 18 || 98 || 1
 +
|-
|-
 +
| 66 || 5
| 66 || 5
 +
|-
|-
 +
| 72 || 3 min
| 72 || 3 min
 +
|-
|-
 +
| 1 ||72 || 10 min
| 1 ||72 || 10 min
 +
|-
|-
 +
| 1 || 4 || inf
| 1 || 4 || inf
 +
|}
|}
 +
 +
'''Results:'''  
'''Results:'''  
 +
* Amplification of DelAE worked
* Amplification of DelAE worked
 +
* bands were cut out and DNA purified using QIAquick Gel Extraction Kit
* bands were cut out and DNA purified using QIAquick Gel Extraction Kit
 +
* sample has to be purified before further use since contamination with propanol was present
* sample has to be purified before further use since contamination with propanol was present
 +
 +
==04-07-2013==
==04-07-2013==
-
===Amplification of FS_02 to FS_05; 11.2 kb===
+
 
 +
===Amplification from FS_02 to FS_05; 11.2 kb===
 +
 
[[File:20130704 DelAF DelFGnew.png|150px|thumb|Gel electrophoresis of the PCR products of DelA-F (first three lane (counted from left)) and DelF-G (4th till 6th lane), 04-07-2013]]   
[[File:20130704 DelAF DelFGnew.png|150px|thumb|Gel electrophoresis of the PCR products of DelA-F (first three lane (counted from left)) and DelF-G (4th till 6th lane), 04-07-2013]]   
 +
[[File:20130704 DelAF DelFG cutnew.png|150px|thumb|Gel after removing DelA-F-fragment, 04-07-2013]]
[[File:20130704 DelAF DelFG cutnew.png|150px|thumb|Gel after removing DelA-F-fragment, 04-07-2013]]
 +
 +
:'''Reaction'''
:'''Reaction'''
 +
{| class="wikitable"
{| class="wikitable"
 +
|-
|-
 +
! what !!  µL 2nd PCR  
! what !!  µL 2nd PCR  
 +
|-
|-
 +
| ''D. acidovorans'' || 1
| ''D. acidovorans'' || 1
 +
|-
|-
 +
| FS_02 (1/10) || 2.5
| FS_02 (1/10) || 2.5
 +
|-
|-
 +
| FS_05 (1/10) || 2.5
| FS_05 (1/10) || 2.5
 +
|-
|-
 +
| Phusion Master Mix || 25
| Phusion Master Mix || 25
 +
|-
|-
 +
| DMSO || 2.5
| DMSO || 2.5
 +
|-
|-
 +
| dd H<sub>2</sub>O || 16.5
| dd H<sub>2</sub>O || 16.5
 +
|}
|}
 +
 +
:'''Conditions'''
:'''Conditions'''
 +
{| class="wikitable"
{| class="wikitable"
 +
|-
|-
 +
! colspan="3" | Biorad MyCycler
! colspan="3" | Biorad MyCycler
 +
|-
|-
 +
! Cycles-PCR !!  temperature [°C] !! Time [s]
! Cycles-PCR !!  temperature [°C] !! Time [s]
 +
|-
|-
 +
| 1 || 98 || 10
| 1 || 98 || 10
 +
|-
|-
 +
| rowspan="3"| 12 || 98 || 1
| rowspan="3"| 12 || 98 || 1
 +
|-
|-
 +
| 68 ↓ 0.5 || 5
| 68 ↓ 0.5 || 5
 +
|-
|-
 +
| 72 || 3 min
| 72 || 3 min
 +
|-
|-
 +
|rowspan="3"| 18 || 98 || 1
|rowspan="3"| 18 || 98 || 1
 +
|-
|-
 +
| 66 || 5
| 66 || 5
 +
|-
|-
 +
| 72 || 3 min
| 72 || 3 min
 +
|-
|-
 +
| 1 ||72 || 10 min
| 1 ||72 || 10 min
 +
|-
|-
 +
| 1 || 4 || inf
| 1 || 4 || inf
 +
|}
|}
 +
 +
'''Results:'''
'''Results:'''
 +
* Amplification of DelAE worked
* Amplification of DelAE worked
 +
* bands were cut out and DNA purified using QIAquick Gel Extraction Kit resulting in a final concentratio of 10ng/µL
* bands were cut out and DNA purified using QIAquick Gel Extraction Kit resulting in a final concentratio of 10ng/µL
 +
* PCR will be repeated and gel slices purified with QIAX II Gel Extraction Kit, which is specifically designed to deliver higher yields when purifying fragments with sizes over 10 kbp
* PCR will be repeated and gel slices purified with QIAX II Gel Extraction Kit, which is specifically designed to deliver higher yields when purifying fragments with sizes over 10 kbp
 +
<div style="clear:both"></div>
<div style="clear:both"></div>

Revision as of 22:48, 29 September 2013

Contents

03-07-2013

Amplification from FS_02 to FS_05; 11.2 kb

File:20130703 DelAF DelFG.png
Amplification of DelA-F and DelF-G, 03-07-13; run at 130 V, 0.8 % gel (TAE)
File:20130703 DelAF DelFG cut.png
Gel after removing the DelA-F-fragment, 03-07-13.
Reaction
µL 1st PCR what µL 2nd PCR
1 D. acidovorans 1
2.5 FS_02 (1/10) 2.5
2.5 FS_05 (1/10) 2.5
25 Phusion Master Mix 25
- DMSO 2.5
19 dd H2O 19


Conditions
Biorad MyCycler
Cycles-PCR temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 3 min
18 98 1
66 5
72 3 min
1 72 10 min
1 4 inf


Results:

  • Amplification of DelAE worked
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit
  • sample has to be purified before further use since contamination with propanol was present


04-07-2013

Amplification from FS_02 to FS_05; 11.2 kb

File:20130704 DelAF DelFGnew.png
Gel electrophoresis of the PCR products of DelA-F (first three lane (counted from left)) and DelF-G (4th till 6th lane), 04-07-2013
File:20130704 DelAF DelFG cutnew.png
Gel after removing DelA-F-fragment, 04-07-2013


Reaction
what µL 2nd PCR
D. acidovorans 1
FS_02 (1/10) 2.5
FS_05 (1/10) 2.5
Phusion Master Mix 25
DMSO 2.5
dd H2O 16.5


Conditions
Biorad MyCycler
Cycles-PCR temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 3 min
18 98 1
66 5
72 3 min
1 72 10 min
1 4 inf


Results:

  • Amplification of DelAE worked
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit resulting in a final concentratio of 10ng/µL
  • PCR will be repeated and gel slices purified with QIAX II Gel Extraction Kit, which is specifically designed to deliver higher yields when purifying fragments with sizes over 10 kbp
Retrieved from "http://2013.igem.org/Team:Heidelberg/Templates/Del_week10_AF"