Team:Heidelberg/Templates/Del week11 G

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Contents

09-07-2013

Amplification from FS_08 to FS_11_short; 6.5 kb

Analytical gel of PCR for amplification of DelG (09.07) and DelOP (reaction 2) (09.07); lane1=Marker, lane2= delG (6.4), lane3=DelOP (2.7kbp); run at 100 V, 0.8 % gel (TAE)
Reaction
what µl
D. acidovorans DSM-39 1
FS_08 (1/10) 1
FS_11_short (1/10) 1
Phusion Master Mix 10
dd H2O 6
DMSO 1
Conditions
Biorad MyCycler
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 2:30
24 98 1
63 5
72 2:30
1 72 10min
1 4 inf

Results:

  • A weak band was visible at the right height.
  • Band was cut out and DNA purified using QIAquick Gel Extration Kit.
  • Concentration after gel extration was too low
  • Maybe increasing the temperature further will result in higher yield.

13-07-2013

Amplification from FS_10 to FS_11s; 3.3 kb

PCR for amplification of Del G (13.07;FS10-FS11short)+ Gel extraction of FS_06 to FS_09 (13.07); lane1=Gel extraction of FS_06 to FS_09 (room temp.), lane2=Gel extraction of FS_06 to FS_09 (50°C), lane3=Del G (13.07;FS10-FS11short), lane4=log2 ladder, lane5 see Indigoidine, lane6=Gel extraction of FS_06 to FS_09 (room temp.); run at 100 V, 0.8 % gel (TAE)
2µl per lane of Gel extracted fragment FS06 to FS09 (13.07); lane3=log2 marker, lane4=tube1-6, lane5=tube7-12
Reaction
what µl
D. acidovorans DSM-39 1
FS_10: (1/10) 2
FS_11_short: (1/10) 2
Phusion flash Master Mix 10
dd H2O 4
DMSO 1
Conditions
Biorad MyCycler
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
70 5
72 1:10 min
1 72 5 min
1 12 inf

Results:

  • No band was visible on the gel.
  • The PCR conditions of the 09-07-2013 should be further optimized.

Amplification from FS_10 to FS_11(s); 3.3 kb

PCR for amplification of Del G (13.07;FS10-FS11); run at 100 V, 0.8 % gel (TAE)
Gel extracted fragements FS10-FS11short and FS10-FS11long (each 1µl); run at 100 V, 0.8 % gel (TAE)
Reaction
what µl
D. acidovorans DSM-39 1
FS_10: (1/10) 2
FS_11 (short or long): (1/10) 2
Phusion flash Master Mix 10
dd H2O 4
DMSO 1
Conditions
Biorad MyCycler
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 1:10 min
18 98 1
66 5
72 1:10 min
1 72 5 min
1 12 inf


Results:

  • Bright bands were visible in both, the PCR with the short and the long primer.
  • The PCR with the short primers worked better than the one with the long primer.
  • Bands were cut out and DNA purified using QIAquick Gel Extration Kit.
  • Concentration after gel extraction with primer FS_11short=6ng/µl in 18µl
  • Concentration after gel extraction with primer FS_11long=4ng/µl in 18µl
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