Team:Heidelberg/Templates/Del week13 AG

From 2013.igem.org

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==27-07-2013==
==27-07-2013==
===Amplification from FS_02 to FS_26; 16.7 kb ===
===Amplification from FS_02 to FS_26; 16.7 kb ===

Latest revision as of 13:28, 1 October 2013

Contents

27-07-2013

Amplification from FS_02 to FS_26; 16.7 kb

2 x Amplification of FS02 to FS_23 27.07.13 200nM/400nM, 4x 2 x Amplification of FS02 to FS_26 27.07.13; run at 100V
Reaction
what µl
D. acidovorans DSM-39 1
FS_02: (1/10) 2
FS_26: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biorad MyCycler*
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
58 5
72 3:50
1 72 12 min
1 10 inf

Results:

  • Amplification of DelAG did not work, unspecific band at approximately 3.5 kbp occured

Amplification 1 from FS_02 to FS_23; 22.8 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

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_23: (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 6:40
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 6:40 min
18 98 1
66 5
72 6:40 min
1 72 10 min
1 4 inf

Results:

  • Amplification of DelAG did not work, several unspecific bands occured

Amplification 2 from FS_02 to FS_23; 22.8 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)

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_23: (1/10) 4/2
Phusion flash Master Mix 10
DMSO 1
dd H2O 0/4
Conditions I
Biorad MyCycler, Biometra TProfessional Basic and Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
60 5
72 6:40
1 72 12 min
1 10 inf

Results:

  • Amplification of DelAG did not work, independent of the cycler only unspecific bands at 4.0 and 5.2 kbp occured
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