Team:Heidelberg/Templates/Del week15 AF

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Contents

07-08-2013

Amplification I from FS_02 to FS_05; 11.2 kb

Amplifcation of DelAF(I), DelFG, DelG (07.08); run at 100 V, 0.8 % gel (TAE)
Amplifcation of DelAF(I), DelFG, DelG (07.08), cut


Reaction
what µL
D. acidovorans SPH-1 1
FS_02 (1/10) 2.5
FS_05 (1/10) 2.5
Phusion Master Mix 25
DMSO 2.5
dd H2O 16.5
Conditions
Biometra TProfessional Basic
Cycles-PCR temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 3 min
18 98 1
66 5
72 3 min
1 72 10 min
1 4 inf

Results:

  • Amplification of DelAF did not work
  • Repeat amplification in case any errors were made

Amplification II from FS_02 to FS_05; 11.2 kb

Amplifcation of DelG (SR01-FS23), pSB4K5 and DelAFII; run at 100 V, 0.8 % gel (TAE)(07.08)
Amplifcation of DelG (SR01-FS23), pSB4K5 and DelAFII cut; run at 100 V, 0.8 % gel (TAE)(07.08)
Reaction
what µL
D. acidovorans SPH-1 1
FS_02 (1/10) 2.5
FS_05 (1/10) 2.5
Phusion Master Mix 25
DMSO 2.5
dd H2O 16.5
Conditions
Biorad MyCycler*
Cycles-PCR temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 3 min
18 98 1
66 5
72 3 min
1 72 10 min
1 4 inf

Results:

  • Amplification of DelAF did not work as no product appeared, therefore conditions of PCR have to be optimized
  • as extremely long amplicons in the past were usually easier to amplify from agar plate (colony PCR) the same conditions will be tried on the colony as template

08-08-2013

Amplification from FS_02 to FS_05; 11.2 kb

Amplifcation of DelAF and DelG; run at 100 V, 0.8 % gel (TAE)(07.08)


Reaction
what µL
D. acidovorans SPH-1 (from agarplate) 1
FS_02 (1/10) 2.5
FS_05 (1/10) 2.5
Phusion Master Mix 25
DMSO 2.5
dd H2O 16.5
Conditions
Biorad MyCycler*
Cycles-PCR temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 3 min
18 98 1
66 5
72 3 min
1 72 10 min
1 4 inf

Results:

  • Amplification of DelAF did not work as no product appeared, therefore conditions of PCR have to be optimized
  • PCR will be repeated with glycerol stock as template

09-08-2013

Amplification from FS_02 to FS_05; 11.2 kb

Amplifcation of DelOP (I), DelFG and DelL ; run at 100 V, 0.8 % gel (TAE)(09.08)
Amplifcation of DelOP (I), DelFG and DelL ; run at 100 V, 0.8 % gel (TAE)(09.08)


Reaction
what µL 2nd PCR
D. acidovorans SPH-1 (glycerol stock) 1
FS_02 (1/10) 2.5
FS_05 (1/10) 2.5
Phusion Master Mix 25
DMSO 2.5
dd H2O 16.5
Conditions
Biorad MyCycler
Cycles-PCR temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 3 min
18 98 1
66 5
72 3 min
1 72 10 min
1 10 inf

Results:

  • Amplification of DelAF did not work as no product appeared, therefore conditions of PCR have to be optimized
  • PCR will be repeated with a higher annealing temperature as primers might not have bound due to secondary structes
  • PCR will be repeated with lower annealing temperatue as primers might not have bound due to high temperatures

10-08-2013

Amplification from FS_02 to FS_05; 11.2 kb

Amplifcation of DelOP and DelAF ; run at 135 V, 0.8 % gel (TAE)(10.08)
Amplifcation of DelOP and DelAF after cutting ; run at 135 V, 0.8 % gel (TAE)(10.08)


Reaction
Reagent DelAF
Template D.acidovorans SPH-1 colony
Primer fw 2 µL FS_02 4 µL FS_02
Primer rev 2 µL FS_05 4 µL FS_05
Phusion flash Ready Mix 10 µL
dd H2O 1 µL -


Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
58 5
72 3:15
1 72 10 min
1 10 inf

Results:

  • Amplification of DelAF did not work at a temperature of 58°C constant
  • as primers might not have been bound at this low temperatue but neither bound at a temperature of 68°C (touchdown or constant), PCR will be repeated at a temperature of 65°C as touchdown and at constant annealing

Amplification II + III from FS_02 to FS_05; 11.2 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 DelAF II + III
Template D.acidovorans SPH-1 colony
Primer fw 4.5 µL FS_02
Primer rev 4.5 µL FS_05
DMSO 1 µL
Phusion Ready Mix 10 µL
Conditions
Biorad MyCycler
Cycles temperature [°C] DelAF II Time Cycles temperature [°C] DelAF III Time
1 98 10 s 1 98 10 s
30 98 1 s 12 98 1 s
65 ↓ 0.5 5 s
65 5 s 72 3:15 min
18 98 1 s
72 3:15 min 66 5 s
72 3:15 min
1 72 10 min 1 72 10 min
1 10 inf 1 10 inf

Results:

  • Amplification of DelAF from the new strain finally worked, both conditions (65°C constant and 65°C touchdown) led to specific product but the touchdown PCR resulted in a smear
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit
  • PCR will be repeated with a slightly lower temperature with constant annealing to increase yield but avoid the smear which appeared due to the low annealing temperatures used in the last cycles of the touchdown PCR.

11-08-2013

Amplification from FS_02 to FS_05; 11.2 kb

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 DelAF
Template D.acidovorans SPH-1 colony
Primer fw 4.5 µL FS_02
Primer rev 4.5 µL FS_05
Phusion Ready Mix 10 µL


Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
62 5
72 3:15
1 72 10 min
1 10 inf

Results:

  • Amplification of DelAF worked out though again a smear and several other bands appeared
  • bands were cut out very precise to avoid carry over of any unwanted amplicon and DNA purified using QIAquick Gel Extraction Kit
  • gradient PCR will be carried out to determine optimal annealing temperature of primers and thereby obtain product of higher specificity suitable for gibson assembly
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