Team:Heidelberg/Notebook/Killing II/7thweek

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   Home    Team   Overview    Advisors  </a></li>  Undergraduates  </a></li>  University  </a></li>  DKFZ  </a></li>  BioQuant  </a></li>  BioRegion Rhein-Neckar  </a></li> </ul> </li>  Project</a> <ul class="DropDownMenu" id="MB1-DDM1">  Overall Project  </a></li>  Material & Methods  </a> <li><a href="http://2008.igem.org/Team:Heidelberg/Project/Sensing"> Sensing  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Project/Killing_I"> Killing I  - Phages  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Project/Killing_II"> Killing II - Colicin  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Project/Visualization"> Visualization  </a></li> </ul> </li>

<li> <a href="http://2008.igem.org/Team:Heidelberg/Parts" style="color: white">Parts</a> <ul class="DropDownMenu" id="MB1-DDM2"> <li><a href="http://2008.igem.org/Team:Heidelberg/Parts"> Submitted Parts  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Parts/Characterization"> Characterization  </a></li> </ul> </li> <li> <a href="http://2008.igem.org/Team:Heidelberg/Modeling" style="color: white">Modeling</a> <ul class="DropDownMenu" id="MB1-DDM3"> <li><a href="http://2008.igem.org/Team:Heidelberg/Modeling"> Overview  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Modeling/Chemotaxis"> Chemotaxis-Colicin  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Modeling/Phage"> Phage Dynamics model  </a></li> </ul> </li> <li> <a href="http://2008.igem.org/Team:Heidelberg/Notebook/Overview" style="color: white">Notebook</a> <ul class="DropDownMenu" id="MB1-DDM5"> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Sensing_Group/Notebook"> Sensing >  </a> <ul class="SideMenu" id="MB1-DDM2-SM1"> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Sensing_Group/Cloning"> Cloning strategy </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Sensing_Group/Notebook"> Notebook </a></li> </ul> </li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_I/Notebook"> Killing I - Phages >  </a> <ul class="SideMenu" id="MB1-DDM2-SM2"> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_I/Cloning"> Cloning strategy </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_I/Notebook"> Notebook </a></li> </ul> </li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/Notebook"> Killing II - Colicin >  </a> <ul class="SideMenu" id="MB1-DDM2-SM3"> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/Cloning"> Cloning strategy </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/Notebook"> Notebook </a></li> </ul> </li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/visualization"> Visualization  </a> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/material"> Material & Methods  </a> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/team_meetings"> Team Meetings  </a> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/seminar"> Seminar on Synthetic Biology  </a> </ul> </li> <li style="width: 160px"> <a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Project_Overview" style="color: white">Human Practice</a> <ul class="DropDownMenu" id="MB1-DDM4"> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Project_Overview"> Project Overview  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Phips_the_Phage"> Phips the Phage  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Essay"> Essay  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Surveys"> Surveys  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Open_Day"> Open Day  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Nobel_Prize"> Nobel Prize  </a></li> </ul> </li>

<li> <a href="http://2008.igem.org/Team:Heidelberg/Sponsors" style="color: white">Sponsors</a> </li> </ul>

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7th week

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pSB1A3-Receiver-Colicin cloning
25.0 µl Phusion Master Mix 2.5 µl T9002_lux_pR_rv_SpeI_BamHI_RBS 2.5 µl T9002_fw_XbaI 20.0 µl H2O 5   colonies --- 50.0 µl
 * Colony-PCR-Screen of transformation for selection of positive clones: 108 colonies

program: 98 °C   5 min 98 °C  10 sec   | 58 °C  30 sec   | 25 cycles 72 °C  45 sec   | 72 °C   5 min 4 °C  constant
 * Gel of Colony-PCR-Screen: 1% Agarose, 135 V, 30 min [[Image:080915_PCR_controll_screen_pSB1A3-Receiver_small.jpg |500 px | center]]

HisTag cloning of Colicins for purification
25.0 µl Phusion Master Mix 2.5 µl ColE1_prot_fw_BamHI/ColE9_prot_fw_BamHI 2.5 µl ColE1_prot_rv_XmaI/ColE9_prot_rv_HindIII 20.0 µl H2O 5   colonies --- 50.0 µl
 * Results of Transformation: Some colonies were grown on the LB-Ampicilin plates. Possibly some positive clones. Verification by Colony-PCR-Screen.
 * Colony-PCR-Screen of transformation for selection of positive clones: 12 x colE1, 12 x colE9

program: 98 °C   5 min 98 °C  10 sec   | 59 °C  30 sec   | 25 cycles 72 °C  45 sec   | 72 °C   5 min 4 °C  constant 25.0 µl Phusion Master Mix 2.5 µl ColE1_prot_fw_BamHI/ColE9_prot_fw_BamHI 2.5 µl ColE1_prot_rv_XmaI/ColE9_prot_rv_HindIII 20.0 µl H2O 1   colony --- 50.0 µl
 * Gel of Colony-PCR-Screen: 1% Agarose, 135 V, 30 min [[Image:080915_HisTag_colony_PCR_screen_small.jpg |500 px | center]]
 * Colony-PCR-Screen of colony 1-10 colE9His to identify the positive clone

program: 98 °C   5 min 98 °C  10 sec   | 68 °C  30 sec   | 25 cycles 72 °C  45 sec   | 72 °C   5 min 4 °C  constant

25.0 µl Phusion Master Mix 2.5 µl ColE1_prot_fw_BamHI/ColE9_prot_fw_BamHI 2.5 µl ColE1_prot_rv_XmaI/ColE9_prot_rv_HindIII 20.0 µl H2O 1   colony --- 50.0 µl
 * Colony-PCR-Screen of colony 1-10 colE9His to identify the positive clone

program: 98 °C   5 min 98 °C  10 sec   | 68 °C  30 sec   | 25 cycles 72 °C  45 sec   | 72 °C   5 min 4 °C  constant

[back]

pSB1A3-Receiver-Colicin cloning
10.0 µl DNA 3.0 µl H2O 2.0 µl NEBuffer I (NEB) 1.5 µl XbaI (20 000 U/ml, NEB) 1.5 µl SpeI (10 000 U/ml, NEB) 2.0 µl BSA 10x (NEB) --- 20.0 µl 25.0 µl Phusion MasterMix (Finnzymes, NEB) 2.5 µl Primer fw (T9002_Lux_pR_SpeI_BamHI_RBS) 2.5 µl Primer rv (T9002_fw_XbaI) 18.0 µl H2O 2.0 µl DNA-Template --- 50.0 µl
 * Miniprep of ONC: eluted in 30 µl H2O (Qiagen, Miniprepkit)
 * Controldigestion of Minipreps: 2 h -> 37 °C, 15 min -> 65 °C
 * Gel of digestion: 1% Agarose, 37 min, 135V [[Image:080916-control_digestion_pSB1A2-receiver_cloning_small.jpg |500 px | center]]
 * Gelresults:
 * Expected Fragments:
 * pSB1A3 ~2000 bp
 * T9002_without_GFP ~1088 bp
 * The expected fragments were not visible. maybe the cloning was not succesfull.
 * Digestion of Miniprpes with SpeI only for further cloninf, because double digestion with BamHI is not recommed. 2 h -> 37 °C, 10 min -> 65 °C
 * Gel of Digestion: 1% Agarose, 30 min, 135 V [[Image:080916-control_digestion_pSB1A2-receiver_cloning_SpeI_small.jpg |500 px | center]]
 * PCR of Minipreps of "positive" clones: To check if we have positive clones we make PCR of the Minipreps we made.

program: 98 °C 30 sec 98 °C 10 sec     | 58 °C 30 sec     | 25 cycles 72 °C 45 sec     | 72 °C  5 min 4 °C constant 10.0 µl 5x Phusion HF Buffer (Finnzymes, NEB) 1.0 µl 10 mM dNTPs (Invitrogen) 2.5 µl Primer fw (T9002_Lux_pR_SpeI_BamHI_RBS) 2.5 µl Primer rv (T9002_fw_XbaI) 31.5 µl H2O 2.0 µl DNA-Template 0.5 µl Phusion DNA-Polymerase (Finnzymes, NEB) --- 50.0 µl
 * Gel of PCR products: 1% Agarose, 135 V, 30 min [[Image:080916-PCR_of_mini_pSB1A3-rec-cloning_small.jpg |500 px | center]]
 * Gelresults:
 * Expected Fragments:
 * T9002_without_GFP ~1088 bp
 * In each probe there is a fragment of ~1500 bp. We expect that this is a form of undigested plasmid. That means that if the PCR worked properly we have no positive clones. To recheck the Minipreps we try an additional PCR with our old protocoll (no Phusion MasterMix).
 * PCR of Minipreps of "positive" clones: To check if we have positive clones we make PCR of the Minipreps we made.

program: 98 °C 30 sec 98 °C 10 sec     | 59 °C 30 sec     | 25 cycles 72 °C 45 sec     | 72 °C  5 min 4 °C constant

HisTag cloning of Colicins for purification

 * Digestion of Minipreps with BamHI/HindIII for Colicin E1 and BamHI/XmaI for ColicinE9His: We want to select the positivt clones.
 * Gel of Digestion: 1% Agarose, 135 V, 30 min [[Image:080916-control_digestion_Hiscloning_small.jpg |500 px | center]]
 * Gelresults:
 * Expected Fragments ColE1:
 * ~1580 bp (Insert)
 * ~3400 bp (Backbone)
 * Expected Fragments ColE9:
 * ~1580 bp (Insert)
 * ~3400 bp (Backbone)
 * None of the inserts is there. Maybe we have only religated backbone. Or maybe only one enzyme cutted the DNA.
 * PCR of Minipreps to check if there are any positive clones. Programm see pSB1A2-Receiver-Cloning. Primers:
 * Colicin E1:
 * ColE1_prot_fw_BamHI
 * ColE1_kil_prot_rv_SpeI
 * Colicin E9:
 * ColE9_prot_fw_BamHI
 * ColE9_prot_rv_HindIII
 * Gel of ColicinE1 PCR: 1% Agarose, 135V, 30 min [[Image:080916-PCR_of_mini_colE1His_small.jpg |500 px | center]]
 * Gel of ColicinE9 PCR: 1% Agarose, 135V, 30 min [[Image:080916-PCR_of_mini_colE9His_small.jpg |500 px | center]]
 * Gelresults of PCR: There were no psitive bands for each PCR. But we used the wrong reverse primers. Because of that we started a new PCR with the right reverse primers (see protocol at pSB1A3-Receiver cloning)

[back]

pSB1A3-Receiver-Colicin cloning

 * Kontrolgel of PCR (09/16/2008) to select positive clones. [[Image:080917-T9002_without_GFP_PCR_controlgel_small.jpg |500 px | center]]
 * Gelresults: positive clones should contain fragments at 1088 bp. Only the colonies 76-80 & 81 - 85 have a dimm band with this size. This could be a positive clone.
 * Minipreps of these ten cultures: eluted in 30 µl H2O, Qiagen Miniprepkit
 * Send plasmid DNA of colonies 78, 86, 87 & 88 to GATC for sequencing:
 * Inoculation of ONC with Reference_promoter cells (BBa_I20260), Colicin E1 cells, Colicin E9 cells and MG1655 in 10 ml TB media.

HisTag cloning of Colicins for purification

 * Gel of ColE9 (top) ColE1 (bottom left) PCR screen (09/15/2008):1% Agarose, 30 min, 135 V [[Image:080917-pQE30His_PCR_controlgel_small.jpg |500 px | center]]
 * Gel of ColE1 and ColE9 His PCR screen (09/15/2008): 1% Agarose, 30 min, 135 V [[Image:080917-pQE30His_PCR_controlgel_3_small.jpg |500 px | center]]
 * Gel of ColE1 His PCR screen (09/15/2008): 1% Agarose, 30 min, 135 V [[Image:080917-pQE30His_PCR_controlgel_2_small.jpg |500 px | center]]
 * Gelresults: Only dimm bands with the right fragment size. This could be the insert or a supercoiled form of the plasmid.
 * ColE9 His PCR-Screen for selection of positive clones: 1% Agarose, 135 V, 30 min, expected fragment size ~630 bp [[Image:080917-pQE30His_PCR_controlgel_4_small.jpg |500 px | center]]
 * Gelrsults: probes 1, 2, 3, 4, 5, 7 & 8 have bands with the right fragment size.
 * Miniprepes of liquid ONC ColE9 His cultures 1-10 ColE1 His cultures 6-10: 1% Agarose, 135 V, 30 min
 * Controldigestion of ColE1His 1 & 2 with EcoRI & PstI: 1% Agarose, 135 V, 30 min [[Image:080917-pQE30E1His_digestion_controlgel_small.jpg |500 px | center]]
 * Results: fragments are to small. Fragments have the size of the pQE30-vector. -> Religated vector.
 * Probes of ColE9 His cloning were send to GATC for sequencing.

[back]

pSB1A2-Receiver-Colicin cloning
2.5 µl Primer reverse (T9002_Lux_pR_SpeI_BamHI_RBS) 2.5 µl Primer forward (T9002_fw_XbaI) 25.0 µl Phusion MM 18.0 µl H2O 2.0 µl Template DNA --- 50.0 µl
 * Sequencing results:
 * only results from forward primer
 * sequence is from backbone -> cloning was not succesfull
 * maybe the backbone we used was already religated
 * ->new start of cloning
 * PCR of T9002 without GFP:

program: 98 °C   30 sec 98 °C   10 sec | 58 °C   30 sec | 30 cycles 72 °C   45 sec | 72 °C    5 min 4 °C   constant Insert 37.0 µl DNA of Gelex 2.0 µl SpeI 1.0 µl XbaI 5.0 µl NEBuffer 2 10x 5.0 µl BSA 10x --- 50.0 µl
 * Gelextraction of PCR: 0.7% Agarose, 45 min, 135 V [[Image:080918-gelextraction_T9002_wo_GFP_PCR_small.jpg |500 px | center]]
 * Digestion of Insert & Backbone: 1 h -> 37 °C, 10 min -> 65 °C

Backbone 37.0 µl T9002 Maxi (1 µg/µl) 2.0 µl SpeI 1.0 µl XbaI 5.0 µl NEBuffer 2 10x 5.0 µl BSA 10x --- 50.0 µl 34 µl DNA 1 µl SAP-Enzymes (Fermentas) 4 µl SAP-Buffer (Fermentas) - 39 µl 5:1 Insert:Vector 15.0 µl Insert 1.0 µl Vector 2.0 µl T4 DNA Ligase 2.0 µl T4 DNA Ligase Buffer --- 20.0 µl
 * Gelextraction of pSB1A3: 0.7% Agarose, 65 min, 100 V, eluated in 50 µl H2O [[Image:080918-gelextraction_pSB1A2_small.jpg |500 px | center]]
 * Sapping of pSB1A3 backbone:
 * ON-Ligation: 14 h -> 16 °C, 20 min -> 65 °C, forever -> 4 °C

3:1 Insert:Vector 14.5 µl Insert 1.5 µl Vector 2.0 µl T4 DNA Ligase 2.0 µl T4 DNA Ligase Buffer --- 20.0 µl

Colicin-Test with eucaryotic cells

 * HeLa and MCF7 cells were treated with supernatants of prior tests. There was no effect in the first hour. Maybe the supernatant was uneffective because it was freezed.

HisTag cloning of Colicins for purification
2.0 µl pColE1/pColE9-J plasmid 2.5 µl Primer fw(ColE1_prot_fw_BamHI/ColE9_prot_fw_BamHI) 2.5 µl Primer rv(ColE1_prot_rv_HindIII/ColE9_prot_rv_XmaI) 18.0 µl H2O 25.0 µl Phusion MM --- 50.0 µl vector: 39.8 µl pQE30 (30 ng/µl) 0.2 µl HindIII/XmaI 5.0 µl NEBuffer 2/4 10x 5.0 µl BSA 10x --- 50.0 µl
 * Sequencing results:
 * religated pQE-30 -> no positive clones
 * ->restart the cloning
 * PCR of colicin E1/E9 proteins:
 * Gelextraction of PCR: 0,7% Agarose, 50 min, 100 V; eluted in 30 µl H2O, Qiagen Gelextraction Kit [[Image:080918-PCR_of_colicin_proteins_small.jpg |500 px | center]]
 * 1st Digestion of Vectors and inserts with HindIII/XmaI for ColE1/E9 HisTag cloning: 1h -> 37 °C, 10 min -> 65 °C

insert: 10.0 µl colicinE1/E9 0.2 µl HindIII/XmaI 5.0 µl NEBuffer 2/4 10x 5.0 µl BSA 10x 29.8 µl H2O --- 50.0 µl 39.0 µl pQE30 (30 ng/µl) 1.0 µl BamHI 5.0 µl NEBuffer 3 10x 5.0 µl BSA 10x --- 50.0 µl
 * Purification of probes with Qiagen PCR Purification Kit
 * 2nd Digestion of Vectors and inserts with BamHIfor ColE1/E9 HisTag cloning: 1h -> 37 °C, 10 min -> 65 °C

insert: 39.0 µl colicinE1/E9 1.0 µl BamHI 5.0 µl NEBuffer 2/4 10x 5.0 µl BSA 10x 29.8 µl H2O --- 50.0 µl 12.0 µl Insert 4.0 µl Vector 2.0 µl T4 DNA Ligase 2.0 µl T4 DNA Ligase Buffer --- 20.0 µl
 * Gelextraction of digested vector and insert: 0.7% Agarose, 45 min, 100 V [[Image:080918-histag_digestion_gelex_small.jpg |500 px | center]]
 * ON Ligation of pQE-30 vector and colicin inserts: 14 h -> 16 °C, 20 min -> 65 °C, forever -> 4 °C

Other

 * Inoculation of ONC with
 * BBa_I23107 -> medium constitutive promoter
 * BBa_I13600 -> CFP cassette
 * BBa_I13602 -> CFP cassette
 * BBa_B0015 -> Terminator
 * BBa_I0500 -> pBAD/araC
 * BBa_F1610 -> AHL sender part

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pSB1A3-Receiver-Colicin cloning
2x 5 µl DNA 5:1 on 50 µl TOP 10 cells + 200 ml LB -> LB-Amp plates 2x 5 µl DNA 3:1 on 50 µl TOP 10 cells + 200 ml LB -> LB-Amp plates
 * Transformation of pSB1A3-T9002-without-GFP ON-Ligations (Transformation protocol Chris)

HisTag cloning of Colicins for purification
20.0 µl DNA 5.0 µl NEBuffer 3 2.0 µl BamHI 2.0 µl PstI 5.0 µl BSA 10x 16.0 µl H2O --- 50.0 µl 2x 5 µl DNA 5:1 on 50 µl TOP 10 cells + 200 ml LB -> LB-Amp plates
 * Digestion of old ColE9His-pQE-30 cloning with BamHI/PstI to verify that there are no positive clones: colonies 1 + 4; 1h 10min -> 37 °C
 * Transformation of ColE9/E1 His tag cloning ON-Ligations (Transformation protocol Chris)
 * Gel of Digestion: 1% Agarose,135 V, 40 min [[Image:080919-controldigestion_pQE-30_ColE9_small.jpg |500 px | center]]
 * Gelresults: Maybe there is no insert or the enzymes didn't cut.

Sender cloning: pBAD promoter

 * BBa_I0500 ONC of 2008 stock did not grow
 * Inoculation of BBa_I0500 of Glycerolstock 2008 & 2004

Team-Meeting

 * Presentationpreparation for Team-Meeting: Which parts can be submitted to the registry?
 * Presentation:
 * ColicinE1 and E9 as part whole cassette
 * Mutagenesis of ColicinE1 Receiver (EcoRI)

Minipreps

 * Of ONCs, eluted in 30µl H2; Qiagen Miniprepkit

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pSB1A3-Receiver-Colicin cloning
25.0 µl Phusion MasterMix (Finnzymes, NEB) 2.5 µl Primer_fw (pSB_ins_fw) 2.5 µl Primer_rv (pSB_ins_rv) 20.0 µl H2O 5    colonies --- 50.0 µl
 * Colony PCR-Screen of pSB1A2-T9002-GFP:

program: 98 °C  5 min 98 °C  10 sec    | 58 °C  30 sec    | 25 cycles 72 °C  45 sec    | 72 °C  5 min 4 °C  constant
 * Gel of PCR screen: No bands were visible. Retry PCR-Screen with Taq-Polymerase and other primers. [[Image:080920-PCR-screen-T9002-GFP_small.jpg |500 px | center]]

HisTag cloning of Colicins for purification
20.0 µl DNA 5.0 µl NEBuffer 3 2.0 µl BamHI 2.0 µl PstI 5.0 µl BSA 10x 16.0 µl H2O --- 50.0 µl
 * Digestion of former Cloning with ColE9His-pQE30 EcoRI/PstI and BamHI/PstI: 1h 37 °C -> Gel

20.0 µl DNA 5.0 µl NEBuffer EcoRI 2.0 µl EcoRI 2.0 µl PstI 5.0 µl BSA 10x 16.0 µl H2O --- 50.0 µl

20.0 µl DNA 5.0 µl NEBuffer 3 2.0 µl BamHI 5.0 µl BSA 10x 18.0 µl H2O --- 50.0 µl

20.0 µl DNA 5.0 µl NEBuffer EcoRI 2.0 µl EcoRI 5.0 µl BSA 10x 18.0 µl H2O --- 50.0 µl


 * Gel of digestion: 1% Agarose, 135V, 30 min: [[Image:080920-controldigestion_pQE30-His_small.jpg |500 px | center]]
 * Inoculation of ONC of 25 colonies per colicin-pQE30

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pSB1A3-Receiver-Colicin cloning
25.0 µl Taq-Polymerase MasterMix (Fermentas) 2.5 µl Primer_rv (T9002_Lux_pR_SpeI_BamHI_RBS) 2.5 µl Primer_fw (T9002_fw_XbaI) 20.0 µl H2O 5   colonies --- 50.0 µl
 * 2nd PCR Screening of pSB1A2-T9002-GFP cloning

program: 95 °C 3 min 95 °C 1 min  | 54 °C 1 min  | 30 cycles 72 °C 1 min  | 72 °C 10 min 4 °C constant


 * Gel of colony PCR screen: 1% Agarose, 135 V, 30 min [[Image:080921_colony_PCR_screen_pSB1A2-T9002-GFP_small.jpg |500 px | center]]
 * Gelresults: Many bands with the right fragment size. New colony PCR screen with single colonies of colonies 31-45 and 66-75
 * Gel of single colony PCR screen: 1% Agarose, 135 V, 30 min [[Image:080921_single_colony_PCR_screen_pSB1A2-T9002-GFP_small.jpg |500 px | center]]
 * Gelresults: Colonies 31, 35, 36, 39, 40, 42, 45, 66, 69, 71, 72 & 73 have the right fragment size. This could be the positive clones
 * Inoculation of LB-Amp ONC from these colonies

HisTag cloning of Colicins for purification
25.0 µl Taq-Polymerase MasterMix (Fermentas) 2.5 µl Primer_rv 2.5 µl Primer_fw 20.0 µl H2O 5   colonies --- 50.0 µl
 * Colony PCR screen to select positive clones:

program: 95 °C 3 min 95 °C 1 min  | 54 °C 1 min  | 30 cycles 72 °C 1 min  | 72 °C 10 min 4 °C constant

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