Team:Heidelberg/Indigoidine

From 2013.igem.org

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                     <ul class="nav nav-tabs">
                     <ul class="nav nav-tabs">
                       <li class="active"><a href="#a10" data-toggle="tab">Overview</a></li>
                       <li class="active"><a href="#a10" data-toggle="tab">Overview</a></li>
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                       <li><a href="#b10" data-toggle="tab">DelA-E</a></li>
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                       <li><a href="#b10" data-toggle="tab">Lab book</a></li>
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                       <li><a href="#c10" data-toggle="tab">DelA-F</a></li>
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                      <li><a href="#d10" data-toggle="tab">DelA-G</a></li>
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                      <li><a href="#e10" data-toggle="tab">DelE-G</a></li>
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                      <li><a href="#j10" data-toggle="tab">DelF-G</a></li>
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                      <li><a href="#f10" data-toggle="tab">DelG</a></li>
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                      <li><a href="#g10" data-toggle="tab">DelO-P</a></li>
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                      <li><a href="#h10" data-toggle="tab">DelL</a></li>
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                      <li><a href="#i10" data-toggle="tab">pSB4K5</a></li>
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                     </ul>
                     </ul>
                 </div>
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                                </html>{{:Team:Heidelberg/Templates/Del_week10_AF}}<html>
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                                </html>{{:Team:Heidelberg/Templates/Del_week10_AG}}<html>
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                              </html>{{:Team:Heidelberg/Templates/Del_week10_EG}}<html>
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                              </html>{{:Team:Heidelberg/Templates/Del_week10_FG}}<html>
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                              </html>{{:Team:Heidelberg/Templates/Del_week10_G}}<html>
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                              </html>{{:Team:Heidelberg/Templates/Del_week10_OP}}<html>
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                              </html>{{:Team:Heidelberg/Templates/Del_week10_L}}<html>
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                              </html>{{:Team:Heidelberg/Templates/Del_week10_pSB4K5}}<html>
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                     <ul class="nav nav-tabs">
                     <ul class="nav nav-tabs">
                       <li class="active"><a href="#a11" data-toggle="tab">Overview</a></li>
                       <li class="active"><a href="#a11" data-toggle="tab">Overview</a></li>
-
                       <li><a href="#b11" data-toggle="tab">DelA-E</a></li>
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                       <li><a href="#b11" data-toggle="tab">Lab book</a></li>
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                      <li><a href="#d11" data-toggle="tab">DelA-G</a></li>
 
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                      <li><a href="#e11" data-toggle="tab">DelE-G</a></li>
 
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                      <li><a href="#f11" data-toggle="tab">DelG</a></li>
 
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                      <li><a href="#g11" data-toggle="tab">DelO-P</a></li>
 
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                      <li><a href="#j11" data-toggle="tab">DelL-P</a></li>
 
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                      <li><a href="#h11" data-toggle="tab">DelL</a></li>
 
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                      <li><a href="#i11" data-toggle="tab">pSB4K5</a></li>
 
                     </ul>
                     </ul>
                 </div>
                 </div>
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                                </html>{{:Team:Heidelberg/Templates/Del_week11_AG}}<html>
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                              </html>{{:Team:Heidelberg/Templates/Del_week11_EG}}<html>
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                              </html>{{:Team:Heidelberg/Templates/Del_week11_G}}<html>
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                              </html>{{:Team:Heidelberg/Templates/Del_week11_LP}}<html>
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                              </html>{{:Team:Heidelberg/Templates/Del_week11_OP}}<html>
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                              </html>{{:Team:Heidelberg/Templates/Del_week11_L}}<html>
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                              </html>{{:Team:Heidelberg/Templates/Del_week11_pSB4K5}}<html>
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                     <ul class="nav nav-tabs">
                     <ul class="nav nav-tabs">
                       <li class="active"><a href="#a12" data-toggle="tab">Overview</a></li>
                       <li class="active"><a href="#a12" data-toggle="tab">Overview</a></li>
-
                       <li><a href="#b12" data-toggle="tab">DelA-E</a></li>
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                       <li><a href="#b12" data-toggle="tab">Lab book</a></li>
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                       <li><a href="#e12" data-toggle="tab">DelE-G</a></li>
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                      <li><a href="#f12" data-toggle="tab">DelG</a></li>
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                      <li><a href="#h12" data-toggle="tab">DelL-P</a></li>
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                      <li><a href="#g12" data-toggle="tab">DelO-P</a></li>
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                     </ul>
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                     <ul class="nav nav-tabs">
                     <ul class="nav nav-tabs">
                       <li class="active"><a href="#a13" data-toggle="tab">Overview</a></li>
                       <li class="active"><a href="#a13" data-toggle="tab">Overview</a></li>
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                       <li><a href="#b13" data-toggle="tab">DelA-E</a></li>
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                       <li><a href="#b13" data-toggle="tab">Lab book</a></li>
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                       <li><a href="#c13" data-toggle="tab">DelA-F</a></li>
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                      <li><a href="#d13" data-toggle="tab">DelA-G</a></li>
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                      <li><a href="#e13" data-toggle="tab">DelE-G</a></li>
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                      <li><a href="#g13" data-toggle="tab">DelO-P</a></li>
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                     </ul>
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                                </html>{{:Team:Heidelberg/Templates/Del_week13_AF}}<html>
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                                </html>{{:Team:Heidelberg/Templates/Del_week13_AG}}<html>
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                                </html>{{:Team:Heidelberg/Templates/Del_week13_E}}<html>
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                              </html>{{:Team:Heidelberg/Templates/Del_week13_EG}}<html>
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                              </html>{{:Team:Heidelberg/Templates/Del_week13_G}}<html>
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                     <ul class="nav nav-tabs">
                     <ul class="nav nav-tabs">
                       <li class="active"><a href="#a14" data-toggle="tab">Overview</a></li>
                       <li class="active"><a href="#a14" data-toggle="tab">Overview</a></li>
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                       <li><a href="#b14" data-toggle="tab">DelA-E</a></li>
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                       <li><a href="#b14" data-toggle="tab">Lab book</a></li>
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                      <li><a href="#c14" data-toggle="tab">DelA-F</a></li>
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                      <li><a href="#d14" data-toggle="tab">DelA-G</a></li>
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                      <li><a href="#f14" data-toggle="tab">DelG</a></li>
 
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                      <li><a href="#g14" data-toggle="tab">DelO-P</a></li>
 
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                     </ul>
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                     <ul class="nav nav-tabs">
                     <ul class="nav nav-tabs">
                       <li class="active"><a href="#a15" data-toggle="tab">Overview</a></li>
                       <li class="active"><a href="#a15" data-toggle="tab">Overview</a></li>
-
                       <li><a href="#b15" data-toggle="tab">DelA-E</a></li>
+
                       <li><a href="#b15" data-toggle="tab">Lab book</a></li>
-
                       <li><a href="#c15" data-toggle="tab">DelA-F</a></li>
+
                        
-
                      <li><a href="#d15" data-toggle="tab">DelA-G</a></li>
+
-
                      <li><a href="#e15" data-toggle="tab">DelE-F</a></li>
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-
                      <li><a href="#f15" data-toggle="tab">DelG</a></li>
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-
                      <li><a href="#g15" data-toggle="tab">DelO-P</a></li>
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-
                      <li><a href="#h15" data-toggle="tab">DelL</a></li>
+
-
                      <li><a href="#i15" data-toggle="tab">pSB4K5</a></li>
+
                     </ul>
                     </ul>
                 </div>
                 </div>
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-
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-
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                              </html>{{:Team:Heidelberg/Templates/Del_week15_pSB4K5}}<html>
 
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                     <ul class="nav nav-tabs">
                     <ul class="nav nav-tabs">
                       <li class="active"><a href="#a16" data-toggle="tab">Overview</a></li>
                       <li class="active"><a href="#a16" data-toggle="tab">Overview</a></li>
-
                       <li><a href="#b16" data-toggle="tab">DelA-F</a></li>                       
+
                       <li><a href="#b16" data-toggle="tab">Lab book</a></li>                       
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                       <li><a href="#d16" data-toggle="tab">DelA-G</a></li> 
+
                        
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                      <li><a href="#f16" data-toggle="tab">DelG</a></li>
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                      <li><a href="#i16" data-toggle="tab">pSB4K5</a></li>
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                              </html>{{:Team:Heidelberg/Templates/Del_week16_pSB4K5}}<html>
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                       <li class="active"><a href="#a17" data-toggle="tab">Overview</a></li>
                       <li class="active"><a href="#a17" data-toggle="tab">Overview</a></li>
                                              
                                              
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                       <li><a href="#i17" data-toggle="tab">pSB4K5</a></li>
+
                       <li><a href="#i17" data-toggle="tab">Lab book</a></li>
                     </ul>
                     </ul>
                 </div>
                 </div>
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                       <li class="active"><a href="#a18" data-toggle="tab">Overview</a></li>
                       <li class="active"><a href="#a18" data-toggle="tab">Overview</a></li>
                                              
                                              
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                       <li><a href="#i18" data-toggle="tab">pSB4K5</a></li>
+
                       <li><a href="#i18" data-toggle="tab">Lab book</a></li>
                     </ul>
                     </ul>
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                 </div>
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                       <li class="active"><a href="#a19" data-toggle="tab">Overview</a></li>
                       <li class="active"><a href="#a19" data-toggle="tab">Overview</a></li>
                                              
                                              
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                       <li><a href="#i19" data-toggle="tab">pSB4K5</a></li>
+
                       <li><a href="#i19" data-toggle="tab">Lab book5</a></li>
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                 </div>
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                       <li class="active"><a href="#a20" data-toggle="tab">Overview</a></li>
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                       <li><a href="#i20" data-toggle="tab">pSB4K5</a></li>
+
                       <li><a href="#i20" data-toggle="tab">Lab book</a></li>
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                     </ul>
                 </div>
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                       <li class="active"><a href="#a21" data-toggle="tab">Overview</a></li>
                                              
                                              
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                       <li><a href="#i21" data-toggle="tab">pSB4K5</a></li>
+
                       <li><a href="#i21" data-toggle="tab">Lab book</a></li>
                     </ul>
                     </ul>
                 </div>
                 </div>
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                       <li class="active"><a href="#a22" data-toggle="tab">Overview</a></li>
                       <li class="active"><a href="#a22" data-toggle="tab">Overview</a></li>
                                              
                                              
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                       <li><a href="#i22" data-toggle="tab">pSB4K5</a></li>
+
                       <li><a href="#i22" data-toggle="tab">Lab book</a></li>
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                     </ul>
                 </div>
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                       <li class="active"><a href="#a23" data-toggle="tab">Overview</a></li>
                                              
                                              
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                       <li><a href="#i23" data-toggle="tab">pSB4K5</a></li>
+
                       <li><a href="#i23" data-toggle="tab">Lab book</a></li>
                     </ul>
                     </ul>
                 </div>
                 </div>

Revision as of 00:12, 5 October 2013

Indigoidine. Proving Modularity of NRPS by Shuffling Domains.

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Methods:

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06-07-2013

Amplification from FS_02 to FS_03; 5.3 kb

PCR for amplification of del fragments E-G and A-E, AE+=PCR AE with DMSO, AE-=PCR without DMSO, M= Marker; wanted amplicon sizes are: AE=5.3kb, EG=11.0
Reaction
what µL
D. acidovorans DSM-39 1
FS_02: (1/10) 1
FS_03: (1/10) 1
Phusion Master Mix 10
DMSO 1/-
dd H2O 6/7
PCR for amplification of del fragments AE and EG, cut
Conditions
Biorad MyCycler
Cycles-PCR temperature [°C] Time [s]
1 98 5
12 98 1
66 ↓ 0.5 5
72 2:30 min
18 98 1
63 5
72 2:30 min
1 72 10 min
1 4 inf

Results:

  • Amplification worked with 5% DMSO
  • Repeat Amplification with the same protocol to increase concentration when DNA is extracted from gel slices

08-07-2013

Amplification from FS_02 to FS_03; 5.3 kb

PCR for amplification of del fragments DelAE (08.07), pSB4K5 (08.07) and DelG (07.07); lane1=Marker, lanes2-4=DelAE (5.3kb), lanes5-10=pSB4K5 (4.2kb), lanes11-13:DelG (6.4kb); 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 Master Mix 25
DMSO 2.5
dd H2O 19


PCR for amplification of del fragments DelAE (08.07), pSB4K5 (08.07) and DelG (07.07), cut
Conditions
Biorad MyCycler
Cycles-PCR temperature [°C] Time [s]
1 98 5
12 98 1
66 ↓ 0.5 5
72 2:30 min
18 98 1
63 5
72 2:30 min
1 72 10 min
1 4 inf

Results:

  • Amplification of DelAE worked
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit

19-07-2013

Amplificaction from FS_02 to FS_03; 5.3 kb

+=with DMSO, -=without DMSO; expected amplicon sizes: LP=6.4kb, 8-9=3.1kb, 21-09 and 20-09=8.1kb, 22-13s=2.6kb, AE=5.3kb; run at 100 V, 0.8 % gel (TAE)
Amplifications of 18.07 and 19.07, cut
Reaction

2x ~50 µL

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

Results:

  • Amplification of DelAE did not work since a different cycler was used

20-07-2013

Amplification from FS_02 to FS_03; 5.3 kb

Amplification of AE, OP, FG (20.07); expected amplicon sizes: 21-09(FG)=8.1kb, 22-13s(OP)=2.6kb, AE=5.3kb; 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 5
12 98 1
66 ↓ 0.5 5
72 2:30
18 98 1
63 5
72 2:30
1 72 10min
1 12 inf

Results:

  • Amplification of DelAE worked but a smear occured, therefore bands were cut out carefully and only used for a test restriction digest

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

29-07-2013

Restriction digest of fragment 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 [bp] 2146, 1862, 1306


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

07-08-2013

Sequencing Results

We send the following samples (amplified fragments of D. acidovorans DSM-39 and backbone pSB4K5 from the partsregistry) to GATC for sequencing:

  • AE (FS_02-FS_03) with Primer FS_02
  • AE (FS_02-FS_03) with Primer FS_03

Alignment of DelAE(FS_02 to FS_03) with Primer FS_02

Alignment of DelAE(FS_02 to FS_03) with Primer FS_03

08-08-2013

Amplification from FS_02 to FS_03; 5.3kb

Amplifcation of DelAE and DelEF and DelOP; run at 100 V, 0.8 % gel (TAE)(07.08)
Amplifcation of DelAE and DelEF and DelOP; run at 100 V, 0.8 % gel (TAE)(07.08)
Reaction
what µL
D. acidovorans SPH-1 1
FS_02: (1/10) 4
FS_03: (1/10) 4
Phusion flash Master Mix 10
Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
58 5
72 1:30
1 72 7min
1 12 inf

Results:

  • Amplification of DelAE was repeated successfully with the new strain SPH-1
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit
  • if fragment is used in Gibson assembly the amplification has to be repeated to increase the amount of product

12-08-2013

Amplification from FS_02 to FS_05; 11.2 kb

Amplification of DelAF using gradient PCR, Amplification of DelOP 72°C 2-step; run at 135 V, 0.8 % gel (TAE)(11.08)
Amplification of DelAF using gradient PCR, Amplification of DelOP 72°C 2-step after cutting; run at 135 V, 0.8 % gel (TAE)(11.08)
Reaction
Reagent DelAF
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
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
67.5 - 65.0 (ΔT = 0.5) ↓ 0.5 5
72 3:20
18 98 1
65.5 - 63.0 (ΔT = 0.5) 5
72 3:20
1 72 10min
1 10 inf

Results:

  • Amplification of DelAF worked well, gradient displays an optimal annealing temperature of 65.5°C, which will be used for further amplifications
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit

13-08-2013

Restriction digest of fragment FS_02 to FS_05; 11.2 kb; 12-08-2013 with EcoRI-HF

Incubation at 37°C for 2 hours

what µL
FS_02 to FS_05 (12-08-2013) 20
EcoRI-HF 1
CutSmart Buffer 2.5
dd H2O 1.5

Expected fragment sizes: 2.26kbp; 4.62kbp; 4.32kbp

Results:

  • Weak bands of about 4.6kbp visible, as well as on of about 2.6kbp
  • digest will be repeated using higher concentrations of DNA to clearify results of restriction digest

Amplification from FS_02 to FS_05; 11.2 kb

Amplification of DelAF (FS_02-FS_05); run at 100 V, 0.8 % gel (TAE)(13.08)
Amplification of DelAF (FS_02-FS_05), cut; run at 100 V, 0.8 % gel (TAE)(13.08)
Reaction

1 sample contains 2µL of DMSO

Reagent DelAF
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 T100
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
65.5 ↓ 0.5 5
72 3:20
18 98 1
63.5 5
72 3:20
1 72 10min
1 10 inf

Results:

  • Amplification of DelAF worked though a slight smear occured, therefore PCR will be repeated on the more precise Biometra TProfessional Basic cylcer again
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit

14-08-2013

Concentration measurement (FS_02 to FS_05; 11.2 kb)

Fragment Primer Date PCR Concentration
DelAF FS02-FS05 11-08-2013 ~10 ng/µL
DelAF FS02-FS05 12-08-2013 0 ng/µL
DelAF FS02-FS05 12-08-2013 0 ng/µL

Results:

  • concentrations are not sufficient for gibson assembly
  • PCR will be repeated and gel slices of different reactions will be pooled for one gel extraction using QIAquick Gel Extraction Kit to obtain the concentrations needed for gibson assembly

Restriction digest of fragment FS_02 to FS_05; 11.2 kb; 11-08-2013 with EcoRI-HF

Test restriction digest of fragment FS_02-FS_05 (12-08) with EcoRI; run at 100 V, 0.8 % gel (TAE)(13.08)


Incubation at 37°C for 2 hours 45min

what µL
FS_02 to FS_05 (11-08-2013) 18
EcoRI-HF 1
CutSmart Buffer 2.5
dd H2O 3.5

Expected fragment sizes: 2.26kbp; 4.62kbp; 4.32kbp

Results:

  • One band of about 5kbp and one of 2.5kbp
  • no clear result, digest will be repeated with another enzyme (ClaI), as the enzyme used was beyond expiration date

Restriction digest of fragment FS_02 to FS_05; 11.2 kb; 10-08-2013 with ClaI

Test restriction digest of fragment FS_02-FS_05 (12-08) with ClaI; run at 100 V, 0.8 % gel (TAE)(14.08)

Incubation at 37°C for 2 hours

what µL
FS_02 to FS_05 (10-08-2013) 20
ClaI 1
CutSmart Buffer 2.5
dd H2O 1.5

Expected fragment sizes: 6.9kbp, 4.3kbp

Results:

  • restriction digest displays the expected fragments, therefore amplification of the desired fragment can be assumed
  • PCR product will be prepared for sequencing by GATC to proof amplification of the desired DNA sequence before Gibson Assembly

17-08-2013

Amplification from FS_02 to FS_05; 11.2 kb

Amplification of DelAF (17-08); run at 100 V, 0.8 % gel (TAE)
Amplification of DelAF, cut (17-08); run at 100 V, 0.8 % gel (TAE)
Reaction of DelAF

6x20 µL

Reagent DelAF
Template D.acidovorans SPH-1 colony
Primer fw 2.5 µL FS_02
Primer rev 2.5 µL FS_05
Phusion Ready Mix 10 µL
DMSO 1 µL
dd H2O 4 µL
Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
65.5 ↓ 0.5 5
72 3:20
18 98 1
63.5 5
72 3:20
1 72 10min
1 10 inf

Results:

  • Amplification of DelAF worked
  • bands were cut out, pooled and DNA purified using QIAquick Gel Extraction Kit to obtain concentrations needed for gibson assembly

18-08-2013

Amplification from FS_02 to FS_05; 11.2 kb

Amplification of DelAF (18-08); run at 100 V, 0.8 % gel (TAE)
Amplification of DelAF, cut (18-08); run at 100 V, 0.8 % gel (TAE)
Reaction of DelAF

6x20 µL

Reagent DelAF
Template D.acidovorans SPH-1 colony
Primer fw 2.5 µL FS_02
Primer rev 2.5 µL FS_05
DMSO 1 µL
Phusion Ready Mix 10 µL
dd H2O 4 µL
Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
65.5 ↓ 0.5 5
72 3:20
18 98 1
63.5 5
72 3:20
1 72 10min
1 10 inf

Results:

  • Amplification of DelAF worked
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit

19-08-2013

Concentration measurement

The concentration of the gel purified, and DpnI digested fragments was measured using a NanoDrop Instrument.

Fragment Primer Date PCR Concentration
pSB4K5 DpnI digested FS_01-FS_16 17-08-2013 101.2 ng/µl
pSB4K5 DpnI digested FS_01-FS_16 18-08-2013 159.2 ng/µl

19-08-2013

Concentration measurement

The concentration of the gel purified, and DpnI digested fragments was measured using a NanoDrop Instrument.

Fragment Primer Date PCR Concentration
pSB4K5 DpnI digested FS_01-FS_16 17-08-2013 101.2 ng/µl
pSB4K5 DpnI digested FS_01-FS_16 18-08-2013 159.2 ng/µl

19-08-2013

Concentration measurement

The concentration of the gel purified, and DpnI digested fragments was measured using a NanoDrop Instrument.

Fragment Primer Date PCR Concentration
pSB4K5 DpnI digested FS_01-FS_16 17-08-2013 101.2 ng/µl
pSB4K5 DpnI digested FS_01-FS_16 18-08-2013 159.2 ng/µl

19-08-2013

Concentration measurement

The concentration of the gel purified, and DpnI digested fragments was measured using a NanoDrop Instrument.

Fragment Primer Date PCR Concentration
pSB4K5 DpnI digested FS_01-FS_16 17-08-2013 101.2 ng/µl
pSB4K5 DpnI digested FS_01-FS_16 18-08-2013 159.2 ng/µl

19-08-2013

Concentration measurement

The concentration of the gel purified, and DpnI digested fragments was measured using a NanoDrop Instrument.

Fragment Primer Date PCR Concentration
pSB4K5 DpnI digested FS_01-FS_16 17-08-2013 101.2 ng/µl
pSB4K5 DpnI digested FS_01-FS_16 18-08-2013 159.2 ng/µl

19-08-2013

Concentration measurement

The concentration of the gel purified, and DpnI digested fragments was measured using a NanoDrop Instrument.

Fragment Primer Date PCR Concentration
pSB4K5 DpnI digested FS_01-FS_16 17-08-2013 101.2 ng/µl
pSB4K5 DpnI digested FS_01-FS_16 18-08-2013 159.2 ng/µl

19-08-2013

Concentration measurement

The concentration of the gel purified, and DpnI digested fragments was measured using a NanoDrop Instrument.

Fragment Primer Date PCR Concentration
pSB4K5 DpnI digested FS_01-FS_16 17-08-2013 101.2 ng/µl
pSB4K5 DpnI digested FS_01-FS_16 18-08-2013 159.2 ng/µl

Thanks to

Retrieved from "http://2013.igem.org/Team:Heidelberg/Indigoidine"