Team:Heidelberg/Templates/Del week13 AE

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Contents

26-07-2013

Restriction digest of fragment FS_02 to FS_03; 5.3 kb; 08-07-2013 with EcoRI-HF

Restriction digest of DelAE (FS_02 to FS_03 ;08.07), DelAF (FS_02 to FS_05; 04.07) and DelOP (FS_22 to FS_13short ;21.07) with EcoRI-HF and digest of DelG (FS_08 to FS_23 cut1 ;23.07) with BglII; run at 100 V, 0.8 % gel (TAE)

Incubation at 37°C for 1 h 45 min

what µL
FS_02 to FS_03 (08-07-2013) 15
EcorRI-HF 0.5
Buffer CutSmart 2
dd H2O 2
Expected fragment lengths [bp] 3054, 2260


Results:

  • restriction digest of DelAE did not work, since incubation time might have been to short

27-07-2013

Amplificaction from FS_02 to FS_03; 5.3 kb

Amplification of DelAE (FS02 to FS03; 27.07,1), Amplification of DelAF (FS_02 to FS05; 27.07); run at 100 V, 0.8 % gel (TAE)
Amplification of DelAE (FS02 to FS03; 27.07,1), Amplification of DelAF (FS_02 to FS05; 27.07), cut; run at 100 V, 0.8 % gel (TAE)
Reaction
what µL
D. acidovorans DSM-39 1
FS_02: (1/10) 2.5
FS_03: (1/10) 2.5
Phusion flash Master Mix 25
DMSO 2.5
dd H2O 19


Conditions
Biorad MyCycler
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
66 ↓ 0.5 5
72 2:30
18 98 1
63 5
72 2:30
1 72 12 min
1 12 inf

Results:

  • Amplification of DelAE didnt work out, only smear occured
  • repeat PCR with better cycler

Amplificaction from FS_02 to FS_03; 5.3 kb

2nd Amplification of DelAE (FS02 to FS03; 27.07,1), first two lanes with 5 µL primer, second two lanes with 2.5 µL primer; run at 100 V, 0.8 % gel (TAE)
gel after excision
Reaction
what µL
D. acidovorans DSM-39 1.5/1
FS_02: (1/10) 2.5/5
FS_03: (1/10) 2.5/5
Phusion flash Master Mix 25
DMSO 2.5
dd H2O 19/14
Conditions
Biorad MyCycler*
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
66 ↓ 0.5 5
72 2:30
18 98 1
63 5
72 2:30
1 72 12 min
1 12 inf

Results:

  • Amplification of DelAE worked with both 200 and 400 nM of Primers, nevertheless amplification was more specific with the higher primer concentration
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit

Amplification I from FS_02 to FS_24; 7.1 kb

Amplifikation of DelFG (FS02-FS24), ; run at 100 V, 0.8 % gel (TAE)

4 reactions, 2 with 200nM Primers and 2 with 400nM Primers (both concentrations for each condition)

Reaction
what µl
D. acidovorans DSM-39 1
FS_02: (1/10) 4/2
FS_24: (1/10) 4/2
Phusion flash Master Mix 10
DMSO 1
dd H2O 0/4
Conditions I
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
65 5
72 3:50
1 72 12 min
1 10 inf


Conditions II
Biometra TProfessional Basic
Cycles-PCR temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 3:50 min
18 98 1
66 5
72 3:50 min
1 72 10 min
1 4 inf

Results:

  • Amplification of DelAE worked with 200 nM primer concentration at an annealing temperature of 68°C and 400 nM at an annealing temperature of 65°C, the product obtained at 65°C was more specific
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit

Amplification II from FS_02 to FS_24; 7.1 kb

2 x Amplification of FS02 to FS_24 27.07.13 68°C Touchdown 200nM/400nM; 4x Amplification of FS-02 to FS23 27.0713 1 & 2 68°C Touchdown 200nM/400nM; 3. & 4. 65°C const. 200/400 nM, run at 100V


2 reactions, 68°C Touchdown with 200nM Primers and 65°C constant with 400nM Primers

Reaction
what µl
D. acidovorans DSM-39 1
FS_02: (1/10) 2/4
FS_24: (1/10) 2/4
Phusion flash Master Mix 10
DMSO 1
dd H2O 0/4
Conditions I
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
65 5
72 5:40
1 72 12 min
1 10 inf


Conditions II
Biometra TProfessional Basic
Cycles-PCR temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 5:40 min
18 98 1
66 5
72 5:40 min
1 72 10 min
1 4 inf

Results:

  • Amplification did not work, neither with 200nM and 68°C touchdown, nor with 400nM and 65°C constant.
  • Repeat amplfication with different conditions as primers did not bind very effectively

Amplification III from FS_02 to FS_24; 7.1 kb

1kbruler / 4xFS02-FS23 60const @T100 200nM; T100 400nM; old 200nM; old 400nM ; red 200nM; red 400nM / FS02-FS24 66td janusA 200nM / FS02-FS24 T100 60const 200nM; 400nM 1kb FS04-FS24.png ; run at 100 V, 0.8 % gel (TAE)
1kbruler / 4xFS02-FS23 60const @T100 200nM; T100 400nM; old 200nM; old 400nM ; red 200nM; red 400nM / FS02-FS24 66td janusA 200nM / FS02-FS24 T100 60const 200nM; 400nM 1kb FS04-FS24.png ; run at 100 V, 0.8 % gel (TAE)

2 reactions, 66°C Touchdown with 200nM Primers and 60°C constant with 400nM Primers

Reaction
what µl
D. acidovorans DSM-39 1
FS_02: (1/10) 2/4
FS_24: (1/10) 2/4
Phusion flash Master Mix 10
DMSO 1
dd H2O 0/4
Conditions I
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
60 5
72 5:40
1 72 12 min
1 10 inf


Conditions II
Biorad C1000 Touch Block A
Cycles-PCR temperature [°C] Time [s]
1 98 10
12 98 1
66 ↓ 0.5 5
72 5:40 min
18 98 1
64 5
72 5:40 min
1 72 10 min
1 4 inf

Results:

  • Amplification from DelAE (7.1 kbp) failed again
  • stick to the old strategy and use previously obtained fragments with different other fragments for gibson assembly.

29-07-2013

Restriction digest of FS_02 to FS_03; 5.3 kb;(27-07-2013; II) with BglII

Test restriction digest (29.07); run at 100 V, 0.8 % gel (TAE)

Incubation at 37°C for about 3 h

what µL
FS_02 to FS_03 (27-07-2013; II) 15
BglII 1
Buffer 3.1 2
dd H2O 2

Expected fragment lengths: 2,146 kb; 1,862 kb; 1,306 kb


Results:

  • Restriction digest shows the expected product sizes
  • indicator for correct amplicon but to be sure, PCR product will be prepared for single read sequencing by GATC
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