Team:Heidelberg/Notebook/Killing II/8thweek

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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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8th week

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go back to the overview

pSB1A3-Receiver-Colicin cloning
25 µl DNA 2 µl SpeI (NEB) 5 µl NEBuffer 2 (NEB) 5 µl BSA 10x (NEB) 13 µl H2O - 50 µl 12 µl DNA 2 µl SpeI (NEB) 2 µl XbaI (NEB) 5 µl BSA 10x (NEB) 5 µl NEBuffer 2 (NEB) 24 µl H2O - 50 µl
 * Digestion of pSB1A3-Receiver Minipreps for identification of positive clones. In addition we want to continue the colicin cloning with the cutted fragment: colonies 31, 35, 36, 39, 40, 42, 45, 66, 69, 71, 72, 73; 1h -> 37 °C
 * Gel of pSB1A3-Receiver Minipreps digestion: 0.7% Agarose, 135 V, 30 min [[Image:080922-pSB1A3-Receiver_Colicin_Cloning_gelex_small.jpg |500 px | center]]
 * Gel result:
 * Expected bands: pSB1A3-Receiver without GFP ~3200 bp
 * There were bands at slightly different heights. We were not able to determine which of these bands have the right size. So we extracted 31, 35, 36, 39, 40, 45, 66, 69, 71, 72 and 73. To identify the right fragments we started a double digest with XbaI and SpeI. Plasmid 42 look like it was not cutted. So we expected that the fragment is ligated reverse into it.
 * Gelextraction: Qiagen Kit, eluted in 40 µl H2O
 * Digestion of pSB1A3-Receiver Minipreps for identification of positive clones: colonies 31, 35, 36, 39, 40, 42, 45, 66, 69, 71, 72, 73; 55 min -> 37 °C

39 µl DNA 1 µl BamHI (NEB) 5 µl NEBuffer 3 (NEB) 5 µl BSA 10x (NEB) - 50 µl
 * Gel of pSB1A3-Receiver Minipreps digestion: 0.7% Agarose, 135 V, 30 min [[Image:080922-pSB1A3-Receiver_Colicin_Cloning_dig_XbaI_SpeI_small.jpg |500 px | center]]
 * Gel results:
 * Expected bands:
 * positive clones: backbone ~2100 bp and insert ~1100 bp, partial digested ~3200
 * religated clones: backbone ~2100 bp
 * reverse inserted clones: no digestion
 * clones with T9002 (with GFP): backbone ~2100 bp and insert (T9002) ~1950 bp
 * 42: reverse inserted. To verify these assumption we send plasmid 42 to GATC for sequencing
 * 31, 39, 40, 45, 69: One thick band at ~2000 bp. In the digestion with SpeI only, these plasmids had the higher bands (~3500 - 4500 bp). Because of these facts we assume that these clones contains T9002 with GFP. To verify this assumption we send plasmid 31 to GATC for sequencing.
 * 35, 36, 66, 71, 72, 73: One band at ~3000. Result looks like the digestion with SpeI only. Maybe these are positive clones and we have lost the XbaI restriction site during the cloning. To verify this assumption we send plasmid 35 to GATC for sequencing.
 * Digestion of Colicin E9, E9lys, E1 insert: 1h 37 °C
 * PCR Purification of Colicin E9, E9lys, E1 insert digestion (Qiagen), eluted in 30 µl

HisTag cloning of Colicins for purification
25.0 µl Taq Master Mic (Fermentas 2.5 µl ColE9_prot_fw_BamHI   2.5 µl ColE9_prot_rv_XmaI 20.0 µl H2O ---
 * Colony-PCR-Screen for selection of positive clones:
 * ColE9: colonies 30 - 37
 * ColE1: colonies 26 - 41

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, 135V, 30 min [[Image:080922_colE9-E1His_PCR_screen_small.jpg |500 px | center]]
 * Gelresults:
 * ColE9His: colonies 32 and 33 have the right fragment size. Inocculation of liquid ONC.
 * ColE1His: There are no colonies with the right fragment size.

[back]

pSB1A3-Receiver-Colicin cloning
2.0 µl template DNA 25.0 µl Taq MasterMix 2.5 µl Primer fw 2.5 µl Primer rv 18.0 µl H2O --- 50.0 µl
 * Minipreps of pSB1A3-T9002-without GFP colonies 31, 35, 36, 39: Qiagen, Miniprepkit
 * Controldigestion of colonies 31 & 35 and BBa_T9002 with BamHI and XbaI and SpeI: 1 h 15 min -> 37 °C
 * Gel of Controldigestion: 1% Agarose, 30 min, 135 V [[Image:080923-control_digestion_BamHI_XbaI_SpeI_small.jpg |500 px | center]]
 * Gelresults:
 * Colony 31: BamHI, XbaI/SpeI and undigested bands have same pattern as BBa_T9002. Maybe this is the luxpR-receiver with GFP.
 * Colony 35: BamHI digestion succesful but Xba and SpeI show only one fragment like it is cutted only once.
 * PCR screening to check if GFP is inside the plasmid (with YFP Primers)

95 °C 1 min 95 °C 1 min   | 56 °C 1 min   | 30 cycles 72 °C 1 min   | 72 °C 10 min 10/20 µl DNA 5.0 µl NEB EcoRI 5.0 µl BSA 10x 1.0 µl EcoRI 1.0 µl PstI 28/18 µl H2O 50.0 µl
 * Gel of PCR: No effect of YFP primers. Maybe the YFP primers binds not correctly. [[Image:080923-PCR_yfp_small.jpg |500 px | center]]
 * Digestion of colonies 31, 35 and BBa_T9002 wit EcoRI/PstI, EcoRI, XbaI, undigested: 1h -> 37 °C
 * Gel of digestion: 1% Agarose, 30 min, 135 V [[Image:080923-control_digestion_XbaI_small.jpg |500 px | center]]
 * Gelresults: Sequencing results have shown that XbaI site is methylated at the GATC pattern. Because of that XbaI is not able to cut. To solve these problem we order new primers with the right prefix.

HisTag cloning of Colicins for purification
18.0 µl H2O 2.5 µl Primer fw 2.5 µl Primer rv 25.0 µl H2O --- 50.0 µl
 * PCR for selection of positive clones:

program 95 °C 30 sec 95 °C  1 min   | 58 °C  1 min   | 25 cycles 72 °C  1 min   | 72 °C 10 min 4 °C constant

General

 * Pascal came home from holiday: Status report

[back]

pSB1A3-Receiver-Colicin cloning
25.0 µl Template DNA 5.0 µl H2O 4.0 µl NEBuffer 2/3 4.0 µl BSA10x 2.0 µl SpeI/BamHI --- 40.0 µl
 * Sequential digestion of T9002-GFP colony 35 and ColE1.4, ColE9.4, ColE9.lys (PCR product) with SpeI and BamHI: 1h 30min -> 37 °C
 * Transformation of ColE1, ColE9 and ColE9lys (Transformation protocoll Chris)

HisTag cloning of Colicins for purification
25.0 µl Template DNA 5.0 µl H2O 4.0 µl NEBuffer 2/3 4.0 µl BSA10x 2.0 µl HindIII/BamHI --- 40.0 µl ColE9His-pQE-30 10.0 µl pQE-30 (2.7 ng/µl) 5.0 µl ColE9His(3.3 ng/µl) 1.0 µl H2O 2.0 µl T4 DNA Lig Buffer 2.0 µl T4 DNA Ligase --- 20.0 µl
 * Sequential digestion of pQE-30 and ColE1 His (PCR product) with HindIII and BamHI: 1st step 1h 30min -> 37 °C Heatinactivation - > PCR-/Gel-Purification -> 2nd step
 * Ligation of old probes

ColE1His-pQE-30 4.5 µl pQE-30 (4.6 ng/µl) 11.5 µl ColE1His(4.4 ng/µl) 2.0 µl T4 DNA Lig Buffer 2.0 µl T4 DNA Ligase --- 20.0 µl

neg controll ColE9His-pQE-30 10.0 µl pQE-30 (2.7 ng/µl) 6.0 µl H2O 2.0 µl T4 DNA Lig Buffer 2.0 µl T4 DNA Ligase --- 20.0 µl

neg controll ColE1His-pQE-30 4.5 µl pQE-30 (4.6 ng/µl) 11.5 µl H2O 2.0 µl T4 DNA Lig Buffer 2.0 µl T4 DNA Ligase --- 20.0 µl

[back]

pSB1A2-Receiver-Colicin cloning
PCR Screening colE1, colE9, colE9lys

colE1: 42 colonies picked (all) colE9 and colE9lys: 48 colonies picked

--> plated on new LB-Amp-Agar plates and PCR:

25,0 µl 2x taq MM (Fermentas) 2,5 µl Primer pSB_insert_fw 2,5 µl Primer colE1_kil_prot_rv_SpeI / colE9_plasmid_rv_SpeI / colE9_lysProt_rv_SpeI 20,0 µl H2O + picked colonies --- 50,0 µl

95 °C 3 min _ 95 °C 1 min | 52 °C 1 min | 28x 72 °C 1 min _| 72 °C 10 min 4 °C for ever

estimated fragment lengths: E1:   3235 bp E9:    3143 bp E9lys: 2167 bp

http://www.igem-heidelberg.de/fileadmin/Results/kill_2/img/080925_PCR_screening_colE1_colE9_colE9lys.jpg

HisTag cloning of Colicins for purification

 * Transformation of the overnight ligation of colE1His and pQE-30 (We, 09/24/202008)

BioBrick sender part

 * To create a sender part which fits BioBrick standards we want to combine BBa_F1610 with an constitutive promoter (BBa_J23107) and in addition wit a pBAD inducible promoter (BBa_I0500). Therefore we cut the promoter parts with SpeI and PstI and the sender gene cassette with XbaI and PstI. The sender gene cassette will be ligated with the cutted promoter parts.

Sender Cloning: pBAD - sender
20.00 µl Miniprep DNA 1.00 µl SpeI (NEB) 0.75 µl PstI (NEB) 5.00 µl NEBuffer 2 5.00 µl BSA 10x 18.25 µl H2O 50.00 µl
 * Digestion of BBa_I0500 with SpeI/PstI: 1h -> 37 °C

20.00 µl Miniprep DNA 1.00 µl XbaI (NEB) 0.75 µl PstI (NEB) 5.00 µl NEBuffer 3 5.00 µl BSA 10x 18.25 µl H2O 50.00 µl 15.0 µl BBa_I0500 8.0 µl BBa_F1610 1.0 µl H2O 3.0 µl T4 DNA Ligase 3.0 µl T4 DNA Ligase Buffer --- 30.0 µl
 * Digestion of BBa_F1610 with XbaI/PstI: 1h -> 37 °C
 * Gelextraction of cutted fragments: qiagen gelextraction kit
 * Ligation of BBa_I0500 with BBa_F1610: 16 °C -> ON

Sender Cloning: constitutive promotor - sender
20.00 µl Miniprep DNA 1.00 µl SpeI (NEB) 0.75 µl PstI (NEB) 5.00 µl NEBuffer 2 5.00 µl BSA 10x 18.25 µl H2O 50.00 µl
 * Digestion of BBa_J23107 with SpeI/PstI: 1h -> 37 °C

20.00 µl Miniprep DNA 1.00 µl XbaI (NEB) 0.75 µl PstI (NEB) 5.00 µl NEBuffer 3 5.00 µl BSA 10x 18.25 µl H2O 50.00 µl 6.0 µl BBa_J23107 16.5 µl BBa_F1610 1.5 µl H2O 3.0 µl T4 DNA Ligase 3.0 µl T4 DNA Ligase Buffer --- 30.0 µl
 * Digestion of BBa_F1610 with XbaI/PstI: 1h -> 37 °C
 * Gelextraction of cutted fragments: qiagen gelextraction kit
 * Ligation of BBa_I0500 with BBa_F1610: 16 °C -> ON

Other

 * Top10 competent cells: preparation of the Top10 cultures (later continued by Kolja and Phillip)

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pSB1A3-Receiver-Colicin cloning
25.0 µl Taq Master Mix (Fermentas) 2.5 µl ColE1_prot_fw_BamHI/ColE9_prot_fw_BamHI/ColE9_prot_fw_BamHI 2.5 µl ColE1_kil_prot_rv_SpeI/ColE9_prot_rv_SpeI/ColE9_lys_prot_rv_SpeI 20.0 µl H2O 5   colonies --- 50.0 µl
 * PCR-Screen of pSB1A3-Receiver-Colicin E1/E9/E9_lys Cloning

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

25.0 µl Taq Master Mix (Fermentas) 2.5 µl ColE1_prot_fw_BamHI 2.5 µl ColE1_kil_prot_rv_SpeI 20.0 µl H2O 5   colonies --- 50.0 µl
 * Gel of E1 and E9 PCR-Screen: 1% Agarose, 135 V, 30 min [[Image:080926_colE9_colE1_colony_PCR_screen_small.jpg |500 px | center]]
 * Gel-Results of E1-PCR-Screen: No colonies with excepted fragment length of ~2100 bp. But positive controll contains also not the right fragment. -> Rerun of PCR-Screen under modified conditions.
 * Gel-Results of E9-PCR-Screen: Colonies 61 - 64 contains excepted fragment with ~1950 bp. -> New PCR-Screen over night of colonies 61-64. In addition: inocculation of liquid ONC of these colonies.
 * Gel of E9lys PCR-Screen: 1% Agarose, 135 V, 30 min [[Image:080926_colE9lys_colony_PCR_screen_small.jpg |500 px | center]]
 * Gel-Results of E9-PCR-Screen: Colonies 1-15 and 21-60 contains excepted fragment with ~1050 bp. -> New PCR-Screen over night of colonies 31-50. In addition: inocculation of liquid ONC of these colonies.
 * Overnight PCR-Screen of pSB1A3-Receiver-Colicin E1 Cloning

program: 95 °C   3 min 95 °C   1 min          | 65 °C   1 min          | 30 cycles 72 °C   2 min          | 72 °C  10 min 4 °C  constant

25.0 µl Taq Master Mix (Fermentas) 2.5 µl ColE9_prot_fw_BamHI/ColE9_prot_fw_BamHI 2.5 µl ColE9_prot_rv_SpeI/ColE9_lys_prot_rv_SpeI 20.0 µl H2O 1   colony(E9 -> 61 - 64, E9lys 31 - 50) --- 50.0 µl
 * Overnight PCR-Screen of pSB1A3-Receiver-Colicin E9/E9lys Cloning

program: 95 °C   3 min 95 °C   1 min          | 54 °C   1 min          | 30 cycles 72 °C   2 min          | 72 °C  10 min 4 °C  constant

HisTag cloning of Colicins for purification
10 µl DNA (32, 33, pQE-30) 5 µl NEBuffer 3 (NEB) 5 µl BSA 10x 2 µl BamHI 2 µl PstI 26 µl H2O - 50 µl
 * Digestion of ColE9His 32 & 33 to verify that there is no colicin insert inside the plasmid. 1 h 37 °C


 * Gel of Digestion: 0.7% Agarose, 135 V, 30 min [[Image:080926_HisTagE9_32_33_controll_digest_small.jpg |500 px | center]]
 * Results of Digestion: No 650 bp fragment visible. In addition the undigested and digested band patterns of colonies 32 & 33 look like the pattern of pQE-30. -> Cloning was not successful

Sender Cloning: pBAD - sender

 * Transformation of Ligation:

Sender Cloning: constitutive promotor - sender

 * Transformation of Ligation

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pSB1A3-Receiver-Colicin cloning

 * Sequencingresults of pSB1A3-Rec cloning:
 * colony 31: contains GFP -> pSB1A3-T9002 with GFP, negative clone
 * colony 35: no results available
 * colony 36: contains no GFP -> positive clone
 * Gel of Colony PCR Screen of pSB1A3-Receiver-ColE9lys cloning: 1% Agarose, 135 V, 30 min [[Image:080927_colE9lys_pSB1A3_Rec_PCR_screen_small.jpg |500 px | center]]
 * Results of Colony PCR Screen of pSB1A3-Receiver-ColE9lys cloning: The colonies 34, 35, 42, 43 and 47 have the right fragment length (1050 bp). Miniprep of these colonies (eluted in 40 µl H2O) and glycerol stocks of colonies 34 and 35.
 * Gel of Colony PCR Screen of pSB1A3-Receiver-ColE1 and ColE9 cloning: 1% Agarose, 135 V, 30 min [[Image:080927_colE1_E9_pSB1A3_Rec_cloning_PCR_screen_small.jpg |500 px | center]]
 * Results of Colony PCR Screen of pSB1A3-Receiver-ColE1 cloning: No colonies with the right fragment length (~2100 bp). -> New cloning
 * Results of Colony PCR Screen of pSB1A3-Receiver-ColE9 cloning: The colonies 61 and 63 have the right fragment length (~1950 bp). Miniprep of these colonies (eluted in 40 µl H2O) and glycerol stock of colony 61.

HisTag cloning of Colicins for purification

 * Gel of Colony PCR Screen of Colicin E1 and E9 Histag cloning: 1% Agarose, 135 V, 30 min [[Image:080927-colE1_colE9_Histag_PCR_screen_small.jpg |500 px | center]]
 * Results of HisTag-Colicin E1 Colony-PCR-Screen: No positive clones with the right fragment length (~1650 bp).
 * Results of HisTag-Colicin E9 Colony-PCR-Screen: Many colonies with right fragment length (~650 bp), but the right band is also visible at the negative controll.

Sender Cloning: pBAD - sender

 * Results of Transformation: one colony -> inocculation of liquid culture

Sender Cloning: constitutive promotor - sender

 * Results of Transformation: two colonies -> inocculation of liquid cultures

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pSB1A3-Receiver-Colicin cloning
10 µl DNA 1 µl XbaI (NEB) 1 µl SpeI (NEB) 5 µl BSA 10x (NEB) 5 µl NEBuffer 2(NEB) 28 µl H2O - 50 µl 7,0 µl Col E1.4 (16,5 ng/µl) 8,4 µl pSB1A3-Receiver-35 (4,1 ng/µl) 0,6 µl H2O 2,0 µl T4 DNA Ligation Buffer 2,0 µl T4 DNA Ligase - 20 µl
 * Digestion of Minipreps: 1h 30 min 37 °C; We did not consider the fact that our XbaI site is methylated so that only SpeI was able to cut.
 * Gel of Digestion: 1% Agarose, 135 V, 30 min [[Image:080928_-_pSB1A3_Receiver_Colicin_E9_E9lys_digestion_small.jpg |500 px | center]]
 * Ligation of pSB1A3-Receiver and Col E1: 14h at 16°C, 15 min at 65°C

Sender Cloning: pBAD - sender
10 µl DNA 1 µl EcoRI (NEB) 1 µl PstI (NEB) 5 µl BSA 10x (NEB) 5 µl EcoRI Buffer(NEB) 28 µl H2O - 50 µl
 * Miniprep of ONC (Qiagen, Miniprep Kit)
 * Digestion of Miniprep: 1h 30 min 37 °C
 * Gel of Digestion: 1% Agarose, 135 V, 30 min [[Image:080928-Sender_Promotor_cloning_digestion_small.jpg |500 px | center]]

Sender Cloning: constitutive promotor - sender
10 µl DNA 1 µl EcoRI (NEB) 1 µl PstI (NEB) 5 µl BSA 10x (NEB) 5 µl EcoRI Buffer(NEB) 28 µl H2O - 50 µl
 * Minipreps of ONC (Qiagen, Miniprep Kit)
 * Digestion of Minipreps: 1h 30 min 37 °C
 * Gel of Digestion: 1% Agarose, 135 V, 30 min; see pBAD sender cloning

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