Template:Team:Bonn:NetworkData

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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= "The LOV-ipaA -vinculin system is a combined system for light inducible heterodimerisation. It consists out of a LOV domain, which undergoes conformational changes upon irradiation with blue light, and the ipaA-vinculin hybridization system. This two building blocks have be combined and described by Lungu et al. in 2012. Lungu et al. (2008) where able to measure a 49-fold difference in target binding upon irradiation as compared to the dark state. However, they further modified the system by mutations of the LOV-ipaA construct and successfully weakend the baseline affinity for vinculin (initial design: 3.5 nM to 69 nM; mutant: 2.4 nM to >40µM affinity for vinculin) to reduce the dark state activity. Lov-ipaA-VinD1 is a powerful tool which allows photocontroled complex formation. To establish this system Lungu et al. (2012)<a href=#ref36.1>36.1</a> fused the Ja helix of the LOV Domain to ipaA.To be more precise they used the LOV2 domain from Avena sativa photopropin 1 (AsLOV2), which – as previously shown – can be used to photomodulate the affinity of peptides for their binding partners (see Figure 1). <div class=contant-image><img src=https://static.igem.org/mediawiki/2013/0/02/Bonn_MS_Figure1_LOV.jpg>Figure 1: General design of AsLOV2 fusion proteins (Lungu et al. 2012)<a href=#ref36.1>36.1</a></div> In other studies had been shown that the LOV domain can be fused to entire protein domains, allowing photomodulation of the protein binding. However, they stated that it might be of high importance to bring the LOV domain closer to ipaA, in order to allow photomodulation in this case, because ipaA is only a peptide and thus more flexible than folded domains.Therefore, Lungu et al. (2012)<a href=#ref36.1>36.1</a> identified similar amino acid sequences in the ipaA peptide and the Ja helix of the LOV Domain and used this combined with molecular modeling to create photomodulateable AsLOV2-ipaA (see Figure 2). <div class=contant-image><img src=https://static.igem.org/mediawiki/2013/5/54/Bonn_MS_Figure2_LOV-ipaA.jpg>Figure 2: Light-inducible LOV-ipaA construct (Lungu et al. 2012)<a href=#ref36.1>36.1</a></div>They were able to proof the functionality of the AsLOV2-ipaA system by heterodimerization in yeast (yeast two-hybrid system or Y2H) The yeast two-hybrid system can be used to monitor protein–protein interactions between two proteins. The system is based on a transcription factor, which is split into two separate fragments, called the binding domain (BD) and activating domain (AD). Each domain is fused to one protein and thus only if the proteins interact with each other BD and AD are close enough to initiate the transcription of a reporter gene.The basic principle of the LOV-ipaA & VinD1 system works as follows. In the dark state the fusion product LOV-ipaA is not able to form a complex with vinculin, because LOV blocks ipaA sterically. However, activation of the LOV domain with blue light induces conformational changes in the fused molecule, which results in a movement of the Ja helix with the ipaA away from LOV. Thereby, ipaA becomes accessible for VinD1 and a Complex is formed.The activation is reversible and the entire system can be genetically encoded. This two facts are the main advantages of this system in contrast to other typically used systems, which like for the chemical system for example, are based on in vivo covalently modified peptides, that can be activated by light induced cleavage. Moreover, the protein used are relatively small and thus should interfere as little as possible with the prokaryotic metabolism, the activity change form dark to light state is high, the system is completely genetically encoded and reversible. But, the most important property of this system is that it allows the light-controlled heterodimerisation of the two split variants of sspB, which is necessary for our system.<h2>References</h2><a name=ref36.1>36.1</a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3334866/>Lungu et al. (2012) Designing photoswitchable peptides using the AsLOV2 domain</a>";

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content.type="~~Background~~";

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content.type="Project";

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case 69:

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content.i = 69;

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content.parents=[37];

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content.childs=[];

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content.titleShort = "Methods";

+

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";

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content.type="Project";

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break;

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@@ Line 314: / Line 314: @@
 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= "The LOV-ipaA -vinculin system is a combined system for light inducible heterodimerisation. It consists out of a LOV domain, which undergoes conformational changes upon irradiation with blue light, and the ipaA-vinculin hybridization system. This two building blocks have be combined and described by Lungu et al. in 2012.</br></br> Lungu et al. (2008) where able to measure a 49-fold difference in target binding upon irradiation as compared to the dark state. However, they further modified the system by mutations of the LOV-ipaA construct and successfully weakend the baseline affinity for vinculin (initial design: 3.5 nM to 69 nM; mutant: 2.4 nM to >40µM affinity for vinculin) to reduce the dark state activity. </br></br> Lov-ipaA-VinD1 is a powerful tool which allows photocontroled complex formation. To establish this system Lungu et al. (2012)<sup><a href=#ref36.1>36.1</a></sup> fused the Ja helix of the LOV Domain to ipaA.</br>To be more precise they used the LOV2 domain from Avena sativa photopropin 1 (AsLOV2), which – as previously shown – can be used to photomodulate the affinity of peptides for their binding partners (see Figure 1). </br><div class=contant-image><img src=https://static.igem.org/mediawiki/2013/0/02/Bonn_MS_Figure1_LOV.jpg></br>Figure 1: General design of AsLOV2 fusion proteins (Lungu et al. 2012)<sup><a href=#ref36.1>36.1</a></sup></div> </br>In other studies had been shown that the LOV domain can be fused to entire protein domains, allowing photomodulation of the protein binding. However, they stated that it might be of high importance to bring the LOV domain closer to ipaA, in order to allow photomodulation in this case, because ipaA is only a peptide and thus more flexible than folded domains.</br></br>Therefore, Lungu et al. (2012)<sup><a href=#ref36.1>36.1</a></sup> identified similar amino acid sequences in the ipaA peptide and the Ja helix of the LOV Domain and used this combined with molecular modeling to create photomodulateable AsLOV2-ipaA (see Figure 2). </br><div class=contant-image><img src=https://static.igem.org/mediawiki/2013/5/54/Bonn_MS_Figure2_LOV-ipaA.jpg></br>Figure 2: Light-inducible LOV-ipaA construct (Lungu et al. 2012)<sup><a href=#ref36.1>36.1</a></sup></div></br>They were able to proof the functionality of the AsLOV2-ipaA system by heterodimerization in yeast (yeast two-hybrid system or Y2H) The yeast two-hybrid system can be used to monitor protein–protein interactions between two proteins. The system is based on a transcription factor, which is split into two separate fragments, called the binding domain (BD) and activating domain (AD). Each domain is fused to one protein and thus only if the proteins interact with each other BD and AD are close enough to initiate the transcription of a reporter gene.</br></br>The basic principle of the LOV-ipaA & VinD1 system works as follows. In the dark state the fusion product LOV-ipaA is not able to form a complex with vinculin, because LOV blocks ipaA sterically. However, activation of the LOV domain with blue light induces conformational changes in the fused molecule, which results in a movement of the Ja helix with the ipaA away from LOV. Thereby, ipaA becomes accessible for VinD1 and a Complex is formed.</br></br>The activation is reversible and the entire system can be genetically encoded. This two facts are the main advantages of this system in contrast to other typically used systems, which like for the chemical system for example, are based on in vivo covalently modified peptides, that can be activated by light induced cleavage. Moreover, the protein used are relatively small and thus should interfere as little as possible with the prokaryotic metabolism, the activity change form dark to light state is high, the system is completely genetically encoded and reversible. But, the most important property of this system is that it allows the light-controlled heterodimerisation of the two split variants of sspB, which is necessary for our system.</br></br><h2>References</h2></br><a name=ref36.1>36.1</a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3334866/>Lungu et al. (2012) Designing photoswitchable peptides using the AsLOV2 domain</a>";
-content.type="Background";
+content.type="Project";
 break;
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 break;
+case 69:
+content.i = 69;
+content.parents=[37];
+content.childs=[];
+content.titleShort = "Methods";
+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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