Team:Heidelberg/Templates/Del week2 EG

From 2013.igem.org

Contents

09-07-2013

Amplification from FS_04 to FS_07; 11.1 kb

File:20130709 DelOP.png
PCR for Amplification of DelEG (09.07), lane1=Marker, lane2 DelG=(touchdown), Lane3= DelEG (constant annealing temperature)(11kbp); run at 100 V, 0.8 % gel (TAE), 
 Wrong description (actually OP) -->picture in wrong section
Reaction
what µl
D. acidovorans DSM-39 1
FS_04: (1/10) 1
FS_07: (1/10) 1
Phusion Master Mix 10
DMSO 1
dd H2O 6
Conditions I
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
Conditions II
Biorad MyCycler
Cycles-PCR temperature [°C] Time [s]
1 98 10
5 98 1
68 5
72 3 min
25 98 1
72 5
72 3 min
1 72 10 min
1 4 inf

Results:

  • Amplification of DelEG did not work

11-07-2013

Amplification from FS_04 to FS_11s; 17.5 kb

File:20130712 DelLP DelFG DelEG DelEG(PHII).png
PCR for amplification of DelLP, DelFG and DelEG; lane1=Ladder log2, lane2=delLP(11.07), lane3=DelFG(11.07), lane4=DelEG (Phusion flash, 11.07), lane5=DelEG (Phusion II,11.07), lane6=1kb ladder plus; DelLP=6.4kbp, DelFG=5.3kbp, DelEG=16.4kbp; run at 100 V, 0.8 % gel (TAE)
Reaction
what µl
D. acidovorans DSM-39 1
FS_04: (1/10) 1
FS_11_short: (1/10) 1
Phusion flash Master Mix 10
DMSO 1
dd H2O 6
Conditions
Biorad MyCycler
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 5:30
18 98 1
66 5
72 5:30
1 72 15min
1 12 inf

Results:

  • Amplification of DelEG did not work
  • Experiment will be repeated with NEB Phusion II Polymerase as Phusion II is not provided as mastermix and therefore GC-buffer can be used

Amplification from FS_04 to FS_11; 17.5 kb; Phusion II

File:20130712 DelLP DelFG DelEG DelEG(PHII).png
PCR for amplification of DelLP, DelFG and DelEG; lane1=Ladder log2, lane2=delLP(11.07), lane3=DelFG(11.07), lane4=DelEG (Phusion flash, 11.07), lane5=DelEG (Phusion II,11.07), lane6=1kb ladder plus; DelLP=6.4kbp, DelFG=5.3kbp, DelEG=16.4kbp; run at 100 V, 0.8 % gel (TAE)
Reaction
what µl
D. acidovorans DSM-39 1
FS_04: (1/10) 1
FS_11_short: (1/10) 1
Phusion II 0.2
DNTP 0.4
Buffer 4
DMSO 0.6
dd H2O 11.8
Conditions
Biorad MyCycler
Cycles temperature [°C] Time [s]
1 98 30
12 98 5
68 ↓ 0.5 30
72 8:10
18 98 5
66 30
72 8:10
1 72 15min
1 17 inf

Results:

  • Amplification of DelEG did not work
  • it seems not to be possible to amplify the desired 17 kbp fragent with the chosen primers and the given template, primercombination will be changed in further amplification attempts

14-07-2013

Amplification from FS_04 to FS_07; 11.1 kb

File:20130714 FS04 TO FS07.png
PCR for amplification of DelEG (FS04-FS07, 14.07); lane1=log2 Marker, lane2=DelEG (protocol 68touchdown), lane3=DelEG (protocol 72touchdown); desired amplicon size=11kbp run at 100 V, 0.8 % gel (TAE)
File:20130714 FS04 TO FS07 cut.png
PCR for amplification of DelEG (FS04-FS07, 14.07) after excision; run at 100 V, 0.8 % gel (TAE)
Reaction
what µl
D. acidovorans DSM-39 1
FS_04: (1/10) 2
FS_07: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions I

Cycler incubation room right

Biorad MyCycler
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 3:00 min
18 98 1
66 5
72 3:00 min
1 72 10 min
1 12 inf
Conditions II

Cycler incubation room left

Biorad MyCycler
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
70 ↓ 0.5 5
72 3:00 min
18 98 1
68 5
72 3:00 min
1 72 10 min
1 12 inf

Results:

  • Amplification of DelEG worked with a touchdown PCR starting from 70°C annealing temperature
  • band was cut out and DNA purified using QIAquick Gel Extraction Kit
  • PCR will be repeated to increase the amount of DNA and gather the concentrations necessary for Gibson Assembly


Retrieved from "http://2013.igem.org/Team:Heidelberg/Templates/Del_week2_EG"