Team:Heidelberg/Templates/Del week11 FG

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Contents

10-07-2013

Amplification I from FS_06 to FS_07; 5.2 kb

PCR for amplification of DelOP, DelFG and DelG; +=with DMSO, -=without DMSO, L=both primers are the long primers, S= one Primer is one of the short primers; lane1=Marker, lane2=delOP(04.07,L+), lane3=DelOP(10.07,L+), lane4=DelOP(10.07,L-), lane5=DelOP(10.07,S+), lane6=DelOP(10.07,S-), lane7=DelFG(10.07,+), lane8=DelFG(10.07,-), lane9=DelOP(09.07,S+), lane10=DelG(09.07,S+); expected amplicon sizes: DelOP=2.5kbp, Del DelFG=ca 5kbp, DelG=6.4kbp; run at 100 V, 0.8 % gel (TAE)
Gel after excision


Reaction
what µl
D. acidovorans DSM-39 1
FS_06: (1/10) 1
FS_07: (1/10) 1
Phusion Master Mix 10
DMSO 1/-
dd H2O 6/7


Conditions
Biorad MyCycler
Cycles-PCR temperature [°C] Time [s]
1 98 10
12 98 1
66 ↓ 0.5 5
72 2:10 min
18 98 1
63 5
72 2:10 min
1 72 10 min
1 4 inf

Results:

  • Amplification did not work, not product is visible
  • PCR will be repeated with lower annealing temperature to increase primer binding

Amplification II from FS_06 to FS_07; 5.2 kb

PCR for amplification of DelFG II; lane1= Marker, lane2= touchdown with DMSO, lane3= touchdown without DMSO, lane4=constant anealing with DMSO, lane5= constant without DMSO ; expected amplicon size: DelFG=ca.5kbp; run at 100 V, 0.8 % gel (TAE)


Reaction
what µl
D. acidovorans DSM-39 1
FS_06: (1/10) 1
FS_07: (1/10) 1
Phusion Master Mix 10
DMSO 1
dd H2O 6
Conditions
Biorad MyCycler
Cycles-PCR temperature [°C] Time [s]
1 98 5
30 98 1
58 5
72 2:30 min
1 72 10 min
1 12 inf

Results:

  • Amplification of DelFG did not work
  • PCR will be repeated with higher annealing temperature as reaction might not have worked due to secondary structures of primers

Amplification III from FS_06 to FS_07; 5.2 kb

Reaction
what µl
D. acidovorans DSM-39 1
FS_06: (1/10) 1
FS_07: (1/10) 1
Phusion Master Mix 10
DMSO 1
dd H2O 6
Conditions
Biorad MyCycler
Cycles-PCR temperature [°C] Time [s]
1 98 5
12 98 1
60 ↓ 0.5 5
72 2:30 min
18 98 1
58 5
72 2:30 min
1 72 10 min
1 12 inf

11-07-2013

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)
Gel after excision

Amplification from FS_06 to FS_07; 5.2 kb

Reaction
what µl
D. acidovorans DSM-39 1
FS_06: (1/10) 1
FS_07: (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 2:30
18 98 1
66 5
72 2:30
1 72 10min
1 12 inf

Results:

  • Amplification of DelFG did not work
  • other primers will be designed in order to amplify the desired sequence from D. acidovorans

12-07-2013

Amplification from FS_06 to FS_11(s); 11.6 kb

PCR for amplification of DelFG (12.07;FS6+FS11) and re-amplification of DelLP(12.07;FS13short+FS15short); lane1=Ladder log2, lane2=DelFG(12.07;FS6+FS11long), lane3=DelFG(12.07;FS6+FS11short), lane4=DelLP, lane5=1kb ladder plus; DelLP=6.4kbp, DelEG=11.6 kbp; run at 100 V, 0.8 % gel (TAE)
Reaction
what µl
D. acidovorans DSM-39 1
FS_06: (1/10) 2
FS_11 (long or short): (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions

Attention: 4 cycles were accidently carried out with an elongation time of 3:00 min

Biorad MyCycler
Cycles temperature [°C] Time [s]
1 98 10
11 98 1
73 ↓ 0.5 5
72 5:00
19 98 1
68 5
72 5:00
1 72 10min
1 12 inf

Results:

  • Amplification of DelFG did not work, neither with the short nor with the long version of FS_11, consequently PCR will be repeated with different primers

13-07-2013

Amplification from FS_06 to FS_09; 8.5 kb

PCR for amplification of DelFG (13.07; FS6-FS9); run at 100 V, 0.8 % gel (TAE)
PCR for amplification of DelFG (13.07; FS6-FS9) after excision


Reaction
what µl
D. acidovorans DSM-39 1
FS_06: (1/10) 2
FS_09: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions

a gradient PCR combined with touchdown was carried out. 12 wells, gradient in the annealing temperature from 74°C - 66°C, resulting in a gradient of 73°C - 65°C in the constant program

Biorad MyCycler
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
74 ↓ 0.5 to 65 ↓ 0.5 5
72 3:00 min
18 98 1
73 to 65 5
72 3:00 min
1 72 10 min
1 12 inf

Results:

  • Amplification of DelFG worked, though several other bands occured, indicating low primer specifity
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit
  • final concenctration after QIAquick Nucleotide Removal Kit: 5ng/µl in 18µl

14-07-2013

Re-Amplification from FS_06 to FS_09; 8.5 kb; 13-07-2013)

PCR for amplification of DelFG (14.07) run at 100 V, 0.8 % gel (TAE)
Reaction
what µl
Template of gel extraction (13-07-2013) 1
FS_06: (1/10) 5
FS_09: (1/10) 5
Phusion flash Master Mix 25
DMSO 2.5
dd H2O 11.5
Conditions
Biorad MyCycler
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
71 ↓ 0.5 5
72 3:00 min
18 98 1
70 5
72 3:00 min
1 72 10 min
1 12 inf

Results:

  • Amplification of DelFG did not work, no product was detectable
Retrieved from "http://2013.igem.org/Team:Heidelberg/Templates/Del_week11_FG"