Team:Heidelberg/Templates/Del week16 FG

From 2013.igem.org

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! Reagent || DelFG
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| Template ||  ''D.acidovorans'' SPH-1  colony  
| Template ||  ''D.acidovorans'' SPH-1  colony  

Revision as of 10:56, 1 October 2013

Contents

13-08-2013

Amplification from FS_21 to FS_07; 5.2 kb

Amplifcation of DelFG (13-08); run at 100 V, 0.8 % gel (TAE)
Reaction
Reagent µl
Template D.acidovorans SPH-1 colony
Primer fw 4 µl FS_21
Primer rev 4 µl FS_07
Phusion Ready Mix 10 µl
DMSO 1 µl
dd H2O 1 µl
Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
55 2:00
1 72 10 min
1 10 inf

Results:

  • Amplification of DelFG worked, but amount of DNA was insufficient
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit
  • PCR will be carried out with another cylcer which by experience of the last amplifications led to higher product yields.

15-08-2013

Amplification from FS_21 to FS_26 ; 5.5 kb

Amplification of DelFG and Backbone pSB4K5; run at 100 V, 0.8 % gel (TAE)(14.08)
Amplification of DelFG and Backbone pSB4K5; run at 100 V, 0.8 % gel (TAE)(14.08)
Reaction

(5x20 µl)

what µl
D. acidovorans SPH-1 colony 1
FS_21: (1/10) 2
FS_26: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
60 5
72 2:15
1 72 10 min
1 10 inf

Results:

  • Amplification of DelFG was successful
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit
  • obtained DNA will consequently be used for Gibson Assembly
  • PCR will be repeated to get higher amounts of DNA and increase concentration by purifying several gel slices using the same QIAquick spin column

16-08-2013

Concentration measurement DelFG

all fragments were gel purified, FG were additionally purified with the nucleotide removal kit

Fragment Primer Date PCR Concentration
DelFG FS21-FS26 29-07-2013 61.4 ng/µl
DelFG after nucleotide removal FS21-FS26 29-07-2013 30 ng/µl

Amplification from FS_21 to FS_26 ; 5.5 kb

Amplification of DelFG ; run at 100 V, 0.8 % gel (TAE)(14.08)
Amplification of DelFG ; run at 100 V, 0.8 % gel (TAE)(14.08)
Reaction

(5x20 µl)

what µl
D. acidovorans SPH-1 colony 1
FS_21: (1/10) 2
FS_26: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
60 5
72 2:15
1 72 10 min
1 10 inf

Results:

  • Amplification of DelFG was successful
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit
  • obtained DNA will consequently be used for Gibson Assembly
Retrieved from "http://2013.igem.org/Team:Heidelberg/Templates/Del_week16_FG"