Team:Heidelberg/Templates/Del week15 AG

From 2013.igem.org

Contents

07-08-2013

Amplification from FS_02 to FS_26; 16.7 kb

Amplifcation of DelAG (FS02-FS26), (FS02-FS11), (FS02-FS23); run at 100 V, 0.8 % gel (TAE)(07.08)
Amplifcation of DelAG; run at 100 V, 0.8 % gel (TAE)(07.08)


Reaction
what µl
D. acidovorans SPH-1 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 did not work as expected, nethertheless band was cut out for further amplification with a Re-PCR or test restriction digest.

08-08-2013

Amplification from FS_02 to FS_23; 22.8 kb

Reaction I
what µl
D. acidovorans SPH-1 1
FS_02: (1/10) 4
FS_23: (1/10) 4
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Reaction II
what µl
D. acidovorans SPH-1 1
FS_02: (1/10) 4
FS_13short: (1/10) 4
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
58 5
72 6:40
1 72 12 min
1 10 inf

10-08-2013

Amplification from FS_02 to FS_26; 16.7 kb

Amplifcation of DelAF (02-05) and DelAG (2-26), (2-13), (2-11s) ; run at 135 V, 0.8 % gel (TAE)(10.08)
Amplifcation of DelAF (02-05) and DelAG (2-26), (2-13), (2-11s) after cutting ; run at 135 V, 0.8 % gel (TAE)(10.08)

Reaction

Reagent DelAG
Template D.acidovorans SPH-1 colony
Primer fw 4.5 µl FS_02
Primer rev 4.5 µl FS_26
DMSO 1 µl
Phusion Ready Mix 10 µl

Conditions

Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 5:20
18 98 1
65 5
72 5:20
1 72 10min
1 10 inf

Results:

  • Amplification of DelAG did not work no product was detectable
  • PCR will be repeated with different Primers

Amplification from FS_02 to FS_11_short; 22.8 kb and from FS_02 to FS_23; 22.8 kb

Amplifcation of DelAF (02-05) and DelAG (02-26), (02-13), (02-11s) ; run at 135 V, 0.8 % gel (TAE)(10.08)
Amplifcation of DelAF (02-05) and DelAG (2-26), (2-13), (2-11s) after cutting ; run at 135 V, 0.8 % gel (TAE)(10.08)
Reaction
Reagent DelAG
Template D.acidovorans SPH-1 colony
Primer fw 4.5 µl FS_02 4.5 µl FS_02
Primer rev 4.5 µl FS_11_short 4.5 µl FS_23
DMSO 1 µl
Phusion Ready Mix 10 µl


Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 7:20
18 98 1
65 5
72 7:20
1 72 10min
1 10 inf

Results:

  • Amplification of DelAG did not work no product was detectable, therefore a Re-PCR using the amplification of DelAG from 10-08-13 will be conducted

11-08-2013

Re-PCR from FS_02 to FS_11; 22.8 kb; 10-08-2013

Amplification of DelAF @62°C const., Re-PCR of DelAG with 1:10 and 1:20 deluted Template from 10.08; run at 135 V, 0.8 % gel (TAE)(10.08)
Amplification of DelAF @62°C const., Re-PCR of DelAG with 1:10 and 1:20 deluted Template from 10.08 after cutting; run at 135 V, 0.8 % gel (TAE)(10.08)
Reaction
Reagent DelAG
Template PCR-Product FS02-FS11s from 10-08-2013
Primer fw 4.5 µl FS_02
Primer rev 4.5 µl FS_11
Phusion Ready Mix 10 µl


Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 7:20
18 98 1
65 5
72 7:20
1 72 10min
1 10 inf

Results:

  • Amplification of DelAG did not work, therefore the template used for the Re-PCR presumably was not the desired amplicon
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