Team:Heidelberg/Templates/Del week12 OP

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Contents

17-07-2013

Amplification from FS_22 to FS_13(s); 2.7 kb

lane5=log2 Marker, lane6=68touchdown (FS13_short), lane7=68touchdown (FS13), lane8=72 2-step (FS13_short), lane9=72 2-step (FS13); run at 100 V, 0.8 % gel (TAE)

4 reactions (with long and short primer FS13 and with conditionI and conditionII)

Reaction of DelOP
what µl
D.acidovorans 1
FS_22: (1/10) 2
FS_13 (long or short): (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4


Conditions I of DelOP
Biorad C1000 Touch Block B
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 1:00 min
18 98 1
66 5
72 1:00 min
1 72 5 min
1 12 inf
Conditions II of DelOP
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
72 1:00 min
1 72 5:00 min
1 12 inf

Results:

  • Amplification of DelOP failed again
  • PCR will be repeated, annealing will be carried out 65°C (touchdown) as primer binding seems not to occure at high temperatues as the ones tested in the last amplification attempts

19-07-2013

Amplification from FS_22 to FS_13s; 2.7 kb

+=with DMSO, -=without DMSO; expected amplicon sizes: LP=6.4kbp, 8-9=3.1kbp, 21-09 and 20-09=8.1kbp, 22-13s=2.6kbp, AE=5.3kbp; run at 100 V, 0.8 % gel (TAE)
Amplifications of 18.07 and 19.07, cut

--> reaction mixture with and without DMSO

Reaction
what µl
D. acidovorans DSM-39 1
FS_22: (1/10) 2
FS_13_short: (1/10) 2
Phusion flash Master Mix 10
DMSO 1/-
dd H2O 4/5
Conditions
Biorad MyCycler*
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
65 ↓ 0.5 5
72 1:00
18 98 1
63 5
72 1:00
1 72 5min
1 12 inf

Results:

  • Amplification of DelOP failed again, unexpected bands as well as a smear occured
  • Annealing temperature will be further decreased, to investigate if amplification of the intended product occurs at lower temperatures

20-07-2013

Amplification from FS_22 to FS_13s; 2.7 kb

Amplification of AE, OP, FG (20.07); expected amplicon sizes: 21-09(FG)=8.1kbp, 22-13s(OP)=2.6kbp, AE=5.3kbp; run at 100 V, 0.8 % gel (TAE)
Reaction
what µl
D. acidovorans DSM-39 1
FS_22: (1/10) 2
FS_13_short: (1/10) 2
Phusion flash Master Mix 10
dd H2O 4
Conditions
Biorad C1000 Touch Block B
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
62 ↓ 0.5 5
72 1:00
18 98 1
60 5
72 1:00
1 72 5min
1 12 inf

Results:

  • Amplification of DelOP did not work, only unintented products as well as a slight smear occured
  • Reaction will be carried out again at lower annealing temperature to allow primer binding to the intended sequences

21-07-2013

Amplification from FS_22 to FS_13s; 2.7 kb

Amplification of DelOP and Re-PCR of DelFG (21.07); run at 100 V, 0.8 % gel (TAE)
Amplification of DelOP and Re-PCR of DelFG (21.07) cut; run at 100 V, 0.8 % gel (TAE)

4x 20µl (with, without DMSO; 60touchdown, 72 twostep)

Reaction
what µl
D. acidovorans DSM-39 1
FS_22: (1/10) 2
FS_13_short: (1/10) 2
Phusion flash Master Mix 10
DMSO 1/-
dd H2O 4/5


Conditions I
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 5
12 98 1
60 ↓ 0.5 5
72 1:00
18 98 1
58 5
72 1:00
1 72 5min
1 12 inf
Conditions II
Biorad MyCycler
Cycles temperature [°C] Time [s]
1 98 5
30 98 1
72 1:00 min
1 72 5:00 min
1 12 inf

Results:

  • Amplification of DelOP led to inconclusive results, a very thin band of the intended length as well as several other bands and a smear occured
  • Band were cut out and DNA purified using QIAquick Gel Extraction Kit to verify amplicon in a re-PCR

Re-PCR of DelOP (FS_22 to FS_13s; 2.7 kb; 19-07-2013)

Amplification of FS20-FS07 (FG), Re-PCRs FS08-FS09 (G), FS22-13_short (OP) and FS04-FS07 (all 21.07); - without DMSO, + with DMSO; run at 100 V, 0.8 % gel (TAE)
Amplification of FS20-FS07 (FG), Re-PCRs FS08-FS09 (G), FS22-13_short (OP) and FS04-FS07 (all 21.07) cut2; run at 100 V, 0.8 % gel (TAE)
Reaction
what µl
Fragment FS_22 to FS_13_short (19-07-2013) 1
FS_22: (1/10) 2
FS_13_short: (1/10) 2
Phusion flash Master Mix 10
DMSO -
dd H2O 5
Conditions
Biorad MyCycler*
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
65 ↓ 0.5 5
72 1:00
18 98 1
63 5
72 1:00
1 72 5min
1 12 inf

Results:

  • Amplification of DelOP did not work
  • PCR will be repeated as re-pcr from the previously obtained sample

22-07-2013

Re-PCR of DelOP (FS_22 to FS_13; 2.7 kb; 19-07-2013)

Re-PCR of DelOP (22-13long), Amplification of DelFG (20-9 and 21-9) (22.07); run at 100 V, 0.8 % gel (TAE)
Re-PCR of DelOP (22-13long), Amplification of DelFG (20-9 and 21-9) (22.07) cut; run at 100 V, 0.8 % gel (TAE)
Reaction
what µl
Fragment FS_22 to FS_13_short 19-07-2013) 1
FS_22: (1/10) 2
FS_13_long: (1/10) 2
Phusion flash Master Mix 10
dd H2O 5
Conditions
Biorad MyCycler*
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
65 ↓ 0.5 5
72 1:00
18 98 1
63 5
72 1:00
1 72 5min
1 12 inf

Results:

  • Re-PCR did lead ot a band at the expected size but also a smear and several unexpected bands
  • Re-PCR will be repeated

24-07-2013

Re-PCR of DelOP (FS_22 to FS_13; 2.7 kb; 19-07-2013)

Amplification of DelFG II (FS20 to FS07; 24.07) and DelOP,; run at 100 V, 0.8 % gel (TAE)
Amplification of DelFG II (FS20 to FS07; 24.07) and DelOP; run at 100 V, 0.8 % gel (TAE)

3x20µl

Reaction
what µl
Fragment FS_22 to FS_13_short (19-07-2013) 1
FS_22: (1/10) 2
FS_13_long: (1/10) 2
Phusion flash Master Mix 10
dd H2O 5
Conditions
Biorad MyCycler*
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
65 ↓ 0.5 5
72 1:00
18 98 1
63 5
72 1:00
1 72 5min
1 12 inf

Results:

  • Re-PCR of DelOP did not work, no bands were visible
  • Re-PCR will be repeated
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