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content.parents=[3]

content.childs=[];

+

content.titleShort = "Protein cleavage";

+

content.titleLong = "Protein cleavage";

content.summary= "Proteins can be activated through cleavage of the precursor protein.";

content.text= "One approach to regulate protein activity is to use posttranslational modification, especially by splitting off an inactivating part from a translated pro-peptide. A change in conformation leads to the functional protein. The cleaving can be effected by enzymatic proteolysis, autoproteolysis or surrounding conditions (e.g. pH).<a href='#1.1'>1.1</a> A simple example is the activation of pepsinogen which is caused by a low pH (fig. 1). It is a powerful system to activate proteins, which is frequently used in nature and science. Unfortunately posttranslational cleaving can usually not be reversed. Once activated the inactive pro-form of the Protein cannot be restored.<h2>References</h2><a name='4.1'>1.1</a> <a href='http://www.ncbi.nlm.nih.gov/books/NBK22589/'> Berg JM, Tymoczko JL, Stryer L.: "Biochemistry. 5th Edition". New York: W H Freeman 2002.</a>";

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content.parents=[36,21,12];

content.childs=[38,45,53,69,52,57,65,100];

-

content.titleShort = "~~project~~ summary";

+

content.titleShort = "Project summary";

-

content.titleLong = "~~our~~ project in a nutshell";

+

content.titleLong = "Our project in a nutshell";

-

content.summary= "We engineer a ~~photoswitachble~~ protein degradation system relying on the ClpXP protease system an a LOV domain from avena sativa";

+

content.summary= "We engineer a photoswitchable protein degradation system relying on the ClpXP protease system and a LOV domain from avena sativa";

content.text= "";

content.type="Project";

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content.i = 107;

content.parents=[105];

-

content.titleShort = "~~Day of Action~~ SynBio";

+

content.titleShort = "SynBio Day";

+

content.titleLong = "SynBio Day";

content.summary= "In cooperation with the iGEM teams of Germany also the team of Bonn organized a day of action for synthetic biology. ";

content.text= "<div align='right'><img src='https://static.igem.org/mediawiki/2013/b/b8/BonnAktionstag.JPG' height='260' width='350'></div>In cooperation with the iGEM teams of Germany also the team of Bonn organized a day of action for synthetic biology. At the 7th of September ten of our members met in Bonn downtown to inform the interested civilians of our city about the international genetically engineered machine competition as well as synthetic biology in general and particularly about our project of light inducible degradation of proteins. Therefore we prepared an information booth near the market place, distributed informative leaflets, visualized our ideas in terms of several posters and on top created a survey to examine the people's opinion. <div align='left'><img src='https://static.igem.org/mediawiki/2013/thumb/9/97/BonnAktionstag2.jpg/800px-BonnAktionstag2.jpg' height='260' width='350'></div> At 9 o'clock in the morning we started in front of the LIMES-Institute to arrange the installation of the stand. By car all the needed equipment was transferred to the city center and there assembled under the eyes of the curious townspeople. Two hours later everything was settled and the official part of the day could begin: From 11 until 3 o'clock intrigued city dweller in every range of age stopped by to examine our exhibition walls and to ask questions, which we answered with pleasure. In the end we were surprised about the brisk participation and the lively discussions that aroused, which reflects in the results of the survey, so that we bundled up and left satisfied. Here you can see the summary of our questionaire: 1. Do you know what synthetic biology means? <div align='center'><img src='https://static.igem.org/mediawiki/2013/b/bd/BonnQuestion_1.png' height='260' width='350'> 2. Do you know what the iGEM competition is about? <div align='center'><img src='https://static.igem.org/mediawiki/2013/4/44/BonnQuestion_2.png' height='260' width='350'> 3. How do you rate the ralation between chance and risk of sythetic biology? <div align='center'><img src='https://static.igem.org/mediawiki/2013/5/58/BonnQuestion3.1.png' height='260' width='350'> 4. What are the main reasons for you that speak against the use of synthetic bioloy? <div align='center'><img src='https://static.igem.org/mediawiki/2013/6/62/BonnQuestion_4.png' height='260' width='350'> 5. Do you think it is important to inform the public better about the topic ' snythetic biology'? <div align='center'><img src='https://static.igem.org/mediawiki/2013/a/a6/Question_5.png' height='260' width='350'> All in all we consider the action as a great success as we were able to reduce prejudices and elucidate people about advantages of synthetic biology.";

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content.childs=[];

content.titleShort = "Methods";

-

content.titleLong = "~~all~~ Protokolls and Methods used by iGEM team Bonn 2013";

+

content.titleLong = "All Protokolls and Methods used by iGEM team Bonn 2013";

content.summary= "The LOV-ipaA -vinculin system is a combined system for light inducible heterodimerisation. This powerful tool, which allows photocontroled complex formation was establish by Lungu et al. in 2012.";

content.text= "<a href='#1'>1. 3A – Assembly</a><a href='#2'>2. Agarose preparation</a><a href='#3'>3.Agarose gel casting</a><a href='#4'>4.loading the agarose gel and starting electrophoresis</a><a href='#5'>5. Preparation of Antibiotic stocks</a><a href='#6'>6. Colony PCR</a><a href='#7'>7. Preparation of Glycerol Stocks (iGEM) </a><a href='#8'>8.Plasmid Preparation </a><a href='#8a'>8a. Midi-Prep (Promega)</a><a href='#8b'>8b. Mini-Prep (Promega)</a><a href='#9'>9.Preparation of LB agar plates</a><a href='#10'>10. PCR - Clean-Up (Macherey und Nagel)</a><a href='#11'>11. Preparation of chemocompetent DH5-alpha cells</a><a href='#12'>12. Re-transformation of BioBricks</a><a href='#13'>13. Strand directed Mutagenesis PCR</a><a href='#14'>14. Transformation using Ligation product (in DH5alpha or XL1Blue) </a><h2><a name='1'>1. 3A – Assembly</a></h2><hr>NOTE: Enzymes and buffers were provided by Promega Restriction (50 µl Reaction)- 25 µl Mastermix Restriction-Enzyme Buffer (2x, with BSA)- add 1 µl from every restriction enzyme to 500ng backbone equimolar DNA- fill up to 50 µl dest. water- incubate for 1.5h-3h at 37ºC - inactivate for 20min at 70ºC (no clean-up, if directly used for ligation) Ligation (20 µl Reaction)- 2,0 µl equimolare restriction samples (inserts)- 20ng/1.5 µl backbone- fill up to 17.7 µl with dest. water- incubate 5min at 37ºC- add 2 µl Ligation buffer (10x) and 0.3 µl T4 DNA Ligase - incubate for 3h RT or 15ºC over night - inactivate for 10min at 70ºC<h2><a name='2'>2.Agarose preparation</a></h2><hr>Materials:- Agarose - 500ml bottle- TBE (1x)Procedure:- dissolve 1g agarose in 100ml TBE (1x), resulting in 1% agarose- heat for 2 minutes in a microwave at maximal power- mix- cook until it boils (1 min)- mix carefully2 clues for a successful boiling: 1. no cords, 2. boiling retardation- store in 65ºC incubator<h2><a name='3'>3.Agarose gel casting</a></h2><hr>- assemble the gel chamber (chamber + 2 fences + 1 - 2 gel combs ) under the ethidiumbromid hood- prepare 50ml falcon tube (label it!)- fill 40ml warm agarose in the falcon tube- add 4 µl ethidiumbromid (1:10.000) under the ethidiumbromid hood- mix by inverting 2-3 times- fill agarose in the gel chamber- wait until the gel becomes solid (about 20minutes)<h2><a name='4'>4.loading the agarose gel and starting electrophoresis</a></h2><hr>- add LoadingDye (1:6) to your sample- remove comb and fence from the gel- place agarose gel in the electrophoresis chamber- pipette samples carefully in the pockets (small pockets: up to 20 µl, big pockets: up to 50 µl)- Place lid on the electrophoresis chamber and connect the electrodes to it.- set parameters (high resolution: 120V, 20-30minutes, low resolution: 130V, 15min)- evaluate gel under UV-light<h2><a name='5'>5. Preparation of Antibiotic stocks</a></h2><hr>Ampicillin:- dissolve 100 mg ampicillin in 1 ml dest. water- store at 20 ºCChloramphenicol:- dissolve 18 mg chloramphenicol in 1 ml ethanol- store at 20 ºC<h2><a name='6'>6. Colony PCR</a></h2><hr>- inoculate 10 µl dest. water with colony.- use 1 µl of this water for one reaction:<img src='https://static.igem.org/mediawiki/2013/9/91/Bonn_MS_Methods1.png' width='550px'><h2><a name='7'>7.Preparation of Glycerol Stocks (iGEM)</a></h2><hr>- autoclave glycerol (60%)- add 0,5 ml Glycerol to 1,5 ml cell culture in a cryo tube- mix- shock freeze in liquid nitrogen- store at -80 ºC<h2><a name='8'>8. Plasmid Preparation</a></h2><hr><h2><a name='8a'>8a. Midi-Prep (Promega)</a></h2><hr>- centrifuge 50 ml of liquid cell culture for 10min at 5000g - decant supernatant- resuspend with 3 ml resuspension solution - add 3 ml cell lysis solution and incubate for maximal 3 min at room temperature - add 5 ml neutralization solution - centrifuge for 20 min at 20 ºC, 5000g - vacuum pump lysat through cleaning column into binding column - abolish cleaning column- vacuum pump with 10 ml endotoxin removal wash solution- vacuum pump with 20 ml column wash solution- dry membrane by vacuum- add 600 µl nuclease free water on membrane- centrifuge for 5 min at 1750 g into a fresh tube<h2><a name='8b'>8b. Mini-Prep (Promega)</a></h2><hr>- fill 1,5 ml overnight-culture in a new tube- centrifuge for 30 seconds at maximal speed - decant supernatant- Repeat previous steps 2-5 times (depending on growth density)- resuspend with 600 µl dest water - add 100 µl cell lysis buffer - after 1min (maximum 2min) add 350 µl of neutralization buffer - centrifuge 3min at maximal speed- place mini column in a collection tube and transfer supernatant into PureYield^TM Mini column- centrifuge for 15 sec at maximal speed- add 200 µl Endotoxin Removal Wash- centrifuge for 15 sec at maximal speed- add 400 µl column Wash solution- centrifuge for 30 sec at maximal speed- place mini column in a new tube- add 30 µl elution buffer to the mini column, incubation at RT for 1 min- centrifuge for 15 seconds at maximal speed - store DNA at -20 ºC<h2><a name='9'>9. Preparation of LB agar plates</a></h2><hr>1l LB agar will result in approximately 30 plates- dissolve 15g agar and 20g LB in 1l dest. Water- autoclave- cool down to 60-70ºC - add antibiotics (1:1000) under the laminar airflow cabinet- mix- cast plates (approximately 20ml / plate)- dry for 2h by room temperature- store at 4ºC<h2><a name='10'>10. PCR - Clean-Up (Macherey und Nagel) </a></h2><hr>Gel Extraction: 1. add double amount NTI to gel2. Incubate 3-7minutes at 50ºC and at 1000rpm (until gel is dissolved)- continue with regular Clean-up Protocol (from step 3.) Cleanup: 1. fill up sample with dest. water to 50 µl, if necessary2. add double amount NTI to the sample3. place column in a collection and add transfer solution to the column4. centrifuge 30 seconds at 11.000g and discard flow through 5. add 700 µl NT3 6. centrifuge 30 seconds at 11.000g and discard flow through 7. repeat step 5) and 6)8. centrifuge 1minute at 11.000g9. place column in a new tube and dry column at 70ºC for 5minsmall parts (<1000bp): 9a. place column in a new tube and add 30 µl Elution buffer 9b. incubate 1min at room temperature9c. Centrifuge 1minute at 11.000g Bigger Parts: (>1000bp) 9A. place column in a new tube and add 20 µl Elution buffer 9B. incubate at 70ºC for 5minutes 9C. centrifuge at 50g for 1minute9D. centrifuge at 11.000g for 1minute9E. repeat step 9A. to 9D.<h2><a name='11'>11. Preparation of chemocompetent DH5-alpha cells</a></h2><hr>- Start with 200 ml Overnight culture with OD600 of 0,6-0,8- centrifuge at 4 ºC, 4500 g, 10 minutes - decant supernatant- resuspend with 40 ml inoune transformation buffer - centrifuge at 4 ºC, 4500 g, 10 minutes - decant supernatant- resuspend in 20 ml inoune transformation buffer - add 1,5 ml DMSO- incubate 10 minutes on ice- transfer 100 – 200 µl into precooled tubes- shock freeze in liquid nitrogen<h2><a name='12'> 12. Re-transformation of Bio Bricks</a></h2><hr>- add 10 µl sterile dest water to DNA on plate - incubate for 10 min at RT- take 2 µl, leave rest on plate - store plates at -20 ºC - add the 2 µl DNA solution to 5 µl competent DH5-alpha- incubate for 30 min on ice - heat shock for 45 s at 42 ºC - incubate 3 min on ice - add 250 µl LB medium at 37 ºC - incubate for 45 min at 37 ºC, 800 rpm - plate 300 µl on Agar-plate with appropriate antibiotic - dry 15min at RT- incubate at 37ºC over night<h2><a name='13'> 13. Strand directed Mutagenesis PCR</a></h2><hr>Prepare master mix and add template as follows: <img src='https://static.igem.org/mediawiki/2013/b/b6/Bonn_MS_Methods2.png' width='550px'>- start PCR-Program: 1. initial denaturation 94ºC for 120 seconds 2. Denaturating 94ºC for 30 seconds3. Annealing 94ºC for 30 seconds4. Elongation 68 for 720 seconds5. Repeat step 2) to 4) 12x <h2><a name='14'>14. Transformation using Ligation product (in DH5alpha or XL1Blue) </a></h2><hr>- thaw bacteria on ice - add 2-4 µl Ligation mixture to 50 µl bacteria- incubate 30minutes on ice - heat shock 30-45 seconds (XL1Blue preferably 35 seconds) at 42ºC- incubate 6min on ice- add 250 µl LB medium at 37ºC- incubate for 45minutes at 37ºC, 800rpm - plate 300 µl on appropriate antibiotic- dry 15 minutes at RT- incubate at 37ºC over night";

@@ Line 61: / Line 61: @@
 content.parents=[3]
 content.childs=[];
+content.titleShort = "Protein cleavage";
+content.titleLong = "Protein cleavage";
 content.summary= "Proteins can be activated through cleavage of the precursor protein.";
 content.text= "One approach to regulate protein activity is to use posttranslational modification, especially by splitting off an inactivating part from a translated pro-peptide. A change in conformation leads to the functional protein. The cleaving can be effected by enzymatic proteolysis, autoproteolysis or surrounding conditions (e.g. pH).<sup><a href='#1.1'>1.1</a></br> A simple example is the activation of pepsinogen which is caused by a low pH (fig. 1). </br> It is a powerful system to activate proteins, which is frequently used in nature and science. Unfortunately posttranslational cleaving can usually not be reversed. Once activated the inactive pro-form of the Protein cannot be restored.<h2>References</h2><a name='4.1'>1.1</a> <a href='http://www.ncbi.nlm.nih.gov/books/NBK22589/'> Berg JM, Tymoczko JL, Stryer L.: &quot;Biochemistry. 5th Edition&quot;. New York: W H Freeman 2002.</a>";
@@ Line 315: / Line 317: @@
 content.parents=[36,21,12];
 content.childs=[38,45,53,69,52,57,65,100];
-content.titleShort = "project summary";
+content.titleShort = "Project summary";
-content.titleLong = "our project in a nutshell";
+content.titleLong = "Our project in a nutshell";
-content.summary= "We engineer a photoswitachble protein degradation system relying on the ClpXP protease system an a LOV domain from avena sativa";
+content.summary= "We engineer a photoswitchable protein degradation system relying on the ClpXP protease system and a LOV domain from avena sativa";
 content.text= "";
 content.type="Project";
@@ Line 819: / Line 821: @@
 content.i = 107;
 content.parents=[105];
-content.titleShort = "Day of Action SynBio";
+content.titleShort = "SynBio Day";
+content.titleLong = "SynBio Day";
 content.summary= "In cooperation with the iGEM teams of Germany also the team of Bonn organized a day of action for synthetic biology. ";
 content.text= "<div align='right'><img src='https://static.igem.org/mediawiki/2013/b/b8/BonnAktionstag.JPG' height='260' width='350'></div>In cooperation with the iGEM teams of Germany also the team of Bonn organized a day of action for synthetic biology. </br> At the 7th of September ten of our members met in Bonn downtown to inform the interested civilians of our city about the international genetically engineered machine competition as well as synthetic biology in general and particularly about our project of light inducible degradation of proteins. </br> Therefore we prepared an information booth near the market place, distributed informative leaflets, visualized our ideas in terms of several posters and on top created a survey to examine the people's opinion. </br> <div align='left'><img src='https://static.igem.org/mediawiki/2013/thumb/9/97/BonnAktionstag2.jpg/800px-BonnAktionstag2.jpg' height='260' width='350'></div> At 9 o'clock in the morning we started in front of the LIMES-Institute to arrange the installation of the stand. By car all the needed equipment was transferred to the city center and there assembled under the eyes of the curious townspeople. Two hours later everything was settled and the official part of the day could begin: From 11 until 3 o'clock intrigued city dweller in every range of age stopped by to examine our exhibition walls and to ask questions, which we answered with pleasure. In the end we were surprised about the brisk participation and the lively discussions that aroused, which reflects in the results of the survey, so that we bundled up and left satisfied. Here you can see the summary of our questionaire:<br/> </br> 1. Do you know what synthetic biology means? </br> <div align='center'><img src='https://static.igem.org/mediawiki/2013/b/bd/BonnQuestion_1.png' height='260' width='350'> <br/> </br> </br> 2. Do you know what the iGEM competition is about? </br> <div align='center'><img src='https://static.igem.org/mediawiki/2013/4/44/BonnQuestion_2.png' height='260' width='350'> <br/> </br> 3. How do you rate the ralation between chance and risk of sythetic biology? </br><div align='center'><img src='https://static.igem.org/mediawiki/2013/5/58/BonnQuestion3.1.png' height='260' width='350'> </br>  4. What are the main reasons for you that speak against the use of synthetic bioloy? </br> <div align='center'><img src='https://static.igem.org/mediawiki/2013/6/62/BonnQuestion_4.png' height='260' width='350'> </br> </br> 5. Do you think it is important to inform the public better about the topic ' snythetic biology'?</br> <div align='center'><img src='https://static.igem.org/mediawiki/2013/a/a6/Question_5.png' height='260' width='350'> </br> </br> All in all we consider the action as a great success as we were able to reduce prejudices and elucidate people about advantages of synthetic biology.";
@@ Line 968: / Line 971: @@
 content.childs=[];
 content.titleShort = "Methods";
-content.titleLong = "all Protokolls and Methods used by iGEM team Bonn 2013";
+content.titleLong = "All Protokolls and Methods used by iGEM team Bonn 2013";
 content.summary= "The LOV-ipaA -vinculin system is a combined system for light inducible heterodimerisation. This powerful tool, which allows photocontroled complex formation was establish by Lungu et al. in 2012.";
 content.text= "<b><a href='#1'>1. 3A – Assembly</a></b></br></br><b><a href='#2'>2. Agarose preparation</a></b></br></br><b><a href='#3'>3.Agarose gel casting</a></b></br></br><b><a href='#4'>4.loading the agarose gel and starting electrophoresis</a></b></br></br><b><a href='#5'>5. Preparation of Antibiotic stocks</a></b></br></br><b><a href='#6'>6. Colony PCR</a></b></br></br><b><a href='#7'>7. Preparation of Glycerol Stocks (iGEM) </a></b></br></br><b><a href='#8'>8.Plasmid Preparation </a></b></br></br><b><a href='#8a'>8a. Midi-Prep (Promega)</a></b></br></br><b><a href='#8b'>8b. Mini-Prep (Promega)</a></b></br></br><b><a href='#9'>9.Preparation of LB agar plates</a></b></br></br><b><a href='#10'>10. PCR - Clean-Up (Macherey und Nagel)</a></b></br></br><b><a href='#11'>11. Preparation of chemocompetent DH5-alpha cells</a></b></br></br><b><a href='#12'>12. Re-transformation of BioBricks</a></b></br></br><b><a href='#13'>13. Strand directed Mutagenesis PCR</a></b></br></br><b><a href='#14'>14. Transformation using Ligation product (in DH5alpha or XL1Blue) </a></b></br></br></br></br><h2><a name='1'>1. 3A – Assembly</a></h2><hr><i>NOTE: Enzymes and buffers were provided by Promega </i></br></br><b>Restriction (50 &micro;l Reaction)</b></br>- 25 &micro;l Mastermix Restriction-Enzyme Buffer (2x, with BSA)</br>- add 1 &micro;l from every restriction enzyme to 500ng backbone equimolar DNA</br>- fill up to 50 &micro;l dest. water</br>- incubate for 1.5h-3h at 37&ordm;C </br>- inactivate for 20min at 70&ordm;C (no clean-up, if directly used for ligation) </br></br><b>Ligation (20 &micro;l Reaction)</b></br>- 2,0 &micro;l equimolare restriction samples (inserts)</br>- 20ng/1.5 &micro;l backbone</br>- fill up to 17.7  &micro;l with dest. water</br>- incubate 5min at 37&ordm;C</br>- add 2 &micro;l Ligation buffer (10x) and 0.3 &micro;l T4 DNA Ligase </br>- incubate for 3h RT or 15&ordm;C over night </br>- inactivate for 10min at 70&ordm;C</br></br></br><h2><a name='2'>2.Agarose preparation</a></h2><hr><b>Materials:</b></br>- Agarose </br>- 500ml bottle</br>- TBE (1x)</br></br><b>Procedure:</b></br>- dissolve 1g agarose in 100ml TBE (1x), resulting in 1% agarose</br>- heat for 2 minutes in a microwave at maximal power</br>- mix</br>- cook until it boils (1 min)</br>- mix carefully</br><i>2 clues for a successful boiling: 1. no cords, 2. boiling retardation</i></br>- store in 65&ordm;C incubator</br></br></br><h2><a name='3'>3.Agarose gel casting</a></h2><hr>- assemble the gel chamber (chamber + 2 fences + 1 - 2 gel combs ) under the ethidiumbromid hood</br>- prepare 50ml falcon tube (label it!)</br>- fill 40ml warm agarose in the falcon tube</br>- add 4 &micro;l  ethidiumbromid (1:10.000) under the ethidiumbromid hood</br>- mix by inverting 2-3 times</br>- fill agarose in the gel chamber</br>- wait until the gel becomes solid (about 20minutes)</br></br></br><h2><a name='4'>4.loading the agarose gel and starting electrophoresis</a></h2><hr>- add LoadingDye (1:6) to your sample</br>- remove comb and fence from the gel</br>- place agarose gel in the electrophoresis chamber</br>- pipette samples carefully in the pockets (small pockets: up to 20 &micro;l, big pockets: up to 50 &micro;l)</br>- Place lid on the electrophoresis chamber and connect the electrodes to it.</br>- set parameters (high resolution: 120V, 20-30minutes, low resolution: 130V, 15min)</br>- evaluate gel under UV-light</br></br></br><h2><a name='5'>5. Preparation of Antibiotic stocks</a></h2><hr><b>Ampicillin:</b></br>- dissolve 100 mg ampicillin in 1 ml dest. water</br>- store at 20 &ordm;C</br></br><b>Chloramphenicol:</b></br>- dissolve 18 mg chloramphenicol in 1 ml ethanol</br>- store at 20 &ordm;C</br></br></br><h2><a name='6'>6. Colony PCR</a></h2><hr>- inoculate 10 &micro;l dest. water with colony.</br>- use 1  &micro;l of this water for one reaction:</br><img src='https://static.igem.org/mediawiki/2013/9/91/Bonn_MS_Methods1.png' width='550px'></br></br></br><h2><a name='7'>7.Preparation of Glycerol Stocks (iGEM)</a></h2><hr>- autoclave glycerol (60%)</br>- add 0,5 ml Glycerol to 1,5 ml cell culture in a cryo tube</br>- mix</br>- shock freeze in liquid nitrogen</br>- store at -80 &ordm;C</br></br></br><h2><a name='8'>8. Plasmid Preparation</a></h2><hr><h2><a name='8a'>8a. Midi-Prep (Promega)</a></h2><hr>- centrifuge 50 ml of liquid cell culture for 10min at 5000g </br>- decant supernatant</br>- resuspend with 3 ml resuspension solution </br>- add 3 ml cell lysis solution and incubate for maximal 3 min at room temperature </br>- add 5 ml neutralization solution </br>- centrifuge for 20 min at 20 &ordm;C, 5000g </br>- vacuum pump lysat through cleaning column into binding column </br>- abolish cleaning column</br>- vacuum pump with 10 ml endotoxin removal wash solution</br>- vacuum pump with 20 ml column wash solution</br>- dry membrane by vacuum</br>- add 600  &micro;l nuclease free water on membrane</br>- centrifuge for 5 min at 1750 g into a fresh tube</br></br></br><h2><a name='8b'>8b. Mini-Prep (Promega)</a></h2><hr>- fill 1,5 ml overnight-culture in a new tube</br>- centrifuge for 30 seconds at maximal speed </br>- decant supernatant</br>- Repeat previous steps 2-5 times (depending on growth density)</br>- resuspend with 600  &micro;l dest water </br>- add 100  &micro;l cell lysis buffer </br>- after 1min (maximum 2min) add 350  &micro;l of neutralization buffer </br>- centrifuge 3min at maximal speed</br>- place mini column in a collection tube and transfer supernatant into PureYield^TM Mini column</br>- centrifuge for 15 sec at maximal speed</br>- add 200  &micro;l Endotoxin Removal Wash</br>- centrifuge for 15 sec at maximal speed</br>- add 400  &micro;l column Wash solution</br>- centrifuge for 30 sec at maximal speed</br>- place mini column in a new tube</br>- add 30  &micro;l elution buffer to the mini column, incubation at RT for 1 min</br>- centrifuge for 15 seconds at maximal speed </br>- store DNA at -20 &ordm;C</br></br></br><h2><a name='9'>9. Preparation of LB agar plates</a></h2><hr>1l LB agar will result in approximately 30 plates</br></br>- dissolve 15g agar and 20g LB in 1l dest. Water</br>- autoclave</br>- cool down to 60-70&ordm;C </br>- add antibiotics (1:1000) under the laminar airflow cabinet</br>- mix</br>- cast plates (approximately  20ml / plate)</br>- dry for 2h by room temperature</br>- store at 4&ordm;C</br></br></br><h2><a name='10'>10. PCR - Clean-Up (Macherey und Nagel) </a></h2><hr><b>Gel Extraction: </b></br>1. add double amount NTI to gel</br>2. Incubate 3-7minutes at 50&ordm;C and at 1000rpm (until gel is dissolved)</br>- continue with regular Clean-up Protocol (from step 3.) </br></br><b>Cleanup: </b></br>1. fill up sample with dest. water to 50 &micro;l, if necessary</br>2. add double amount NTI to the sample</br>3. place column in a collection and add transfer solution to the column</br>4. centrifuge 30 seconds at 11.000g and discard flow through </br>5. add 700 &micro;l NT3 </br>6. centrifuge 30 seconds at 11.000g and discard flow through </br>7. repeat step 5) and 6)</br>8. centrifuge 1minute at 11.000g</br>9. place column in a new tube and dry column at 70&ordm;C for 5min</br></br>small parts (<1000bp): </br>9a. place column in a new tube and add 30 &micro;l Elution buffer </br>9b. incubate 1min at room temperature</br>9c. Centrifuge 1minute at 11.000g </br></br>Bigger Parts: (>1000bp) </br>9A. place column in a new tube and add 20 &micro;l Elution buffer </br>9B. incubate at 70&ordm;C for 5minutes </br>9C. centrifuge at 50g for 1minute</br>9D. centrifuge at 11.000g for 1minute</br>9E. repeat step 9A. to 9D.</br></br></br><h2><a name='11'>11. Preparation of chemocompetent DH5-alpha cells</a></h2><hr>- Start with 200 ml Overnight culture with OD<sub>600</sub> of 0,6-0,8</br>- centrifuge at 4 &ordm;C, 4500 g, 10 minutes </br>- decant supernatant</br>- resuspend with 40 ml inoune transformation buffer </br>- centrifuge at 4 &ordm;C, 4500 g, 10 minutes </br>- decant supernatant</br>- resuspend in 20 ml inoune transformation buffer </br>- add 1,5 ml DMSO</br>- incubate 10 minutes on ice</br>- transfer 100 – 200 &micro;l into precooled tubes</br>- shock freeze in liquid nitrogen</br></br></br><h2><a name='12'> 12. Re-transformation of Bio Bricks</a></h2><hr>- add 10  &micro;l sterile dest water to DNA on plate </br>- incubate for 10 min at RT</br>- take 2  &micro;l, leave rest on plate </br>- store plates at -20 &ordm;C </br>- add the 2  &micro;l DNA solution to 5  &micro;l competent DH5-alpha</br>- incubate for 30 min on ice </br>- heat shock for 45 s at 42 &ordm;C </br>- incubate 3 min on ice </br>- add 250  &micro;l LB medium at 37 &ordm;C </br>- incubate for 45 min at 37 &ordm;C, 800 rpm </br>- plate 300 &micro;l on Agar-plate with appropriate antibiotic </br>- dry 15min at RT</br>- incubate at 37&ordm;C over night</br></br></br><h2><a name='13'> 13. Strand directed Mutagenesis PCR</a></h2><hr>Prepare master mix and add template as follows: </br><img src='https://static.igem.org/mediawiki/2013/b/b6/Bonn_MS_Methods2.png' width='550px'></br>- start PCR-Program: </br>1. initial denaturation 94&ordm;C for 120 seconds </br>2. Denaturating 94&ordm;C for 30 seconds</br>3. Annealing 94&ordm;C for 30 seconds</br>4. Elongation 68 for 720 seconds</br>5. Repeat step 2) to 4) 12x </br></br></br><h2><a name='14'>14. Transformation using Ligation product (in DH5alpha or XL1Blue) </a></h2><hr>- thaw bacteria on ice </br>- add 2-4 &micro;l Ligation mixture to 50 &micro;l bacteria</br>- incubate 30minutes on ice </br>- heat shock 30-45 seconds (XL1Blue preferably 35 seconds) at 42&ordm;C</br>- incubate 6min on ice</br>- add 250 &micro;l LB medium at 37&ordm;C</br>- incubate for 45minutes at 37&ordm;C, 800rpm </br>- plate 300 &micro;l on appropriate antibiotic</br>- dry 15 minutes at RT</br>- incubate at 37&ordm;C over night</br>";

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