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| - | <div class="week">
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| - | <h4>Week 7
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| - | <div class="project_Name">
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| - | <h4>Interlab Study
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| - | <h2> 08.12.2014 </h2>
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| - | <br/> The 4 needed parts are: BBa_J23101, BBa_J23115, BBa_E0240 and BBa_I20260. Corresponding wells were located on 2014 Distribution kit plates and resuspended with 10 µL steril water. That permits to obtain a DNA concentration around 0.2 ng/µl (according to the registry. Solutions were transferred into 1 ml eppendorf tubes and stored at -20°C.
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| - | <br/> To amplify fragments, a PCR was performed on the 4 constructions, with the mix described on table 1.
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| - | <a href="URL IMAGE" class="image">
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| - | <img alt="IMAGE" src="https://static.igem.org/mediawiki/2014/a/a8/PCR12082014.jpg" width="400px;" class="thumbimage"/>
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| - | <a href="https://static.igem.org/mediawiki/2014/a/a8/PCR12082014.jpg" class="internal" title="Enlarge">
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| - | <center>Table 1: PCR mix preparation </center>
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| - | <br/> Distribution of 49 µl of mix per PCR tube. Application of program IGEM Q5 PCR.
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| - | <a href="https://static.igem.org/mediawiki/2014/5/5f/Thermocycling_cond_Q5.jpg" class="image">
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| - | <img alt="IMAGE" src="https://static.igem.org/mediawiki/2014/5/5f/Thermocycling_cond_Q5.jpg" width="200px;" class="thumbimage"/>
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| - | </a>
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| - | <a href="https://static.igem.org/mediawiki/2014/5/5f/Thermocycling_cond_Q5.jpg" class="internal" title="Enlarge">
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| - | <img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="Symbol"/>
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| - | <center>Table 2: IGEM Q5 PCR program thermocycling conditions </center>
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| - | Preparation of a 1% agarose gel: 0.56 g of Top Vision agarose (Thermo Scientific) + 50 ml of TAE 1X.
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| - | <br/>Microwave 30s by 30s until agarose total dissolution
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| - | Gel was cooling down until to be lukewarm, one BET drop was added.
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| - | Gel was loaded with 10µl per sample previously added with 2 µl of loading dye 6X, and 5 µl for ladders.
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| - | Gel running 45 minutes at 100 mV in TAE 1X buffer.
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| - | <div class="thumbinner" style="width:502px;">
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| - | <a href="URL IMAGE" class="image">
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| - | <img alt="IMAGE" src="https://static.igem.org/mediawiki/2014/b/bf/Gel12082014.jpg" width="400px;" class="thumbimage"/>
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| - | </a>
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| - | <div class="thumbcaption">
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| - | <a href="https://static.igem.org/mediawiki/2014/b/bf/Gel12082014.jpg" class="internal" title="Enlarge">
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| - | <img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="Symbol"/>
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| - | </a>
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| - | <center>Figure 1: 1% agarose gel of PCR products. <p style="padding-top:1%;">Lane 1: BBa_J23115 PCR product, Lane 2: pBHR1 PCR product, Lane 3 and 6: Purple 2-Log ladder NEB, Lane 4: BBa_E0240 PCR product, Lane 5: BBa_I20260 PCR product, Lane 7: 1 Kb plus Ladder Ogene Ruller and Lane 8: BBa_J23101 PCR product </p></center>
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| - | <br/>
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| - | We expected to obtain one band per PCR sample corresponding to the interesting amplified fragment.
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| - | For Lane 2, 3 and 6 it was ok. We had the expected profile. By contrast 2 bands were visible, one at the expected size (around 1200 bp) and another around 600 bp. We decided to perform a purification on gel of each bands.
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| - | <h2> 08.13.2014 </h2>
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| - | PCR products were cleaned with the GeneJET purification kit (Thermo Scientific).
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| - | <br/> DNA quantification with the NanoDrop 2000 (Thermo Scientific).
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| - | <br/>
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| - | <br/>Preparation of a 1% agarose gel: 0.56 g of Top Vision agarose (Thermo Scientific) + 50 ml of TAE 1X.
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| - | <br/>Microwave 30s by 30s until agarose total dissolution
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| - | Gel was cooling down until to be lukewarm, one BET drop was added.
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| - | Gel was loaded with 10µl per sample previously added with 2 µl of loading dye 6X, and 5 µl for ladders.
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| - | Gel running 45 minutes at 100 mV in TAE 1X buffer.
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| - | <div class="center">
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| - | <div class="thumb tnone">
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| - | <div class="thumbinner" style="width:502px;">
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| - | <a href="/wiki/images/0/0a/Gel13082014.jpg" class="image">
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| - | <img alt="IMAGE" src="/wiki/images/0/0a/Gel13082014.jpg" width="400px;" class="thumbimage"/>
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| - | </a>
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| - | <div class="thumbcaption">
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| - | <div class="magnify">
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| - | <a href="/wiki/images/0/0a/Gel13082014.jpg" class="internal" title="Enlarge">
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| - | <img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="Symbol"/>
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| - | </a>
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| - | </div>
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| - | <center>Figure 2: 1% agarose gel of PCR products from 08.12.2014 after PCR clean up. Lane 1 and 7: Purple 2-Log ladder NEB, Lane 2: BBa_J23101 purified PCR product, Lane 3: BBa_J23115 purified PCR product, Lane 4: BBa_E0240 purified PCR product, Lane 5: BBa_I20260 purified PCR product and Lane 6: pBHR1 purified PCR product </center>
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| - | <br/>
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| - | Profiles were same as before PCR clean up. To sequence the amplified parts, we decided to a purification of fragments from the gel as presented below.
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| - | <br/>Preparation of a 1% agarose gel: 0.56 g of Top Vision agarose (Thermo Scientific) + 50 ml of TAE 1X.
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| - | <br/>Microwave 30s by 30s until agarose total dissolution
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| - | Gel was cooling down until to be lukewarm, one BET drop was added.
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| - | Gel was loaded with 30 µl of BBa_E0240 purified PCR product and BBa_I20260 purified PCR product added with 6 µl of loading dye, at 10µl of the mix per well.
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| - | Gel running 45 minutes at 100 mV in TAE 1X buffer.
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| - | <div class="center">
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| - | <div class="thumb tnone">
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| - | <div class="thumbinner" style="width:502px;">
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| - | <a href="URL IMAGE" class="image">
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| - | <img alt="IMAGE" src="https://static.igem.org/mediawiki/2014/a/ae/Gel13082014bis.jpg" width="300px;" class="thumbimage"/>
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| - | </a>
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| - | <div class="thumbcaption">
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| - | <div class="magnify">
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| - | <a href="https://static.igem.org/mediawiki/2014/a/ae/Gel13082014bis.jpg" class="internal" title="Enlarge">
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| - | <img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="Symbol"/>
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| - | </a>
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| - | <center>Figure 2: 1% agarose gel of PCR products from 08.12.2014 after PCR clean up. Lane 4 and 8: Purple 2-Log ladder NEB, Lane 1, 2 and 3: BBa_E0240 purified PCR product, Lane 5, 6 and 7: BBa_I20260 purified PCR product </center>
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| - | Four pieces of gel were sampled in four tubes to perform a DNA purification from gel.
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| - | <h2> 08.14.2014 </h2>
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| - | To amplify BBa_I20260 and BBa_0240, a PCR was perform with the mix described Table 1. The program IGEM Q5 PCR was applied, see Table 2.
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| - | <br/> PCR products were cleaned with the GeneJET purification kit (Thermo Scientific).
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| - | <br/> DNA quantification with the NanoDrop 2000 (Thermo Scientific).
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| - | <br/>Preparation of a 1% agarose gel: 0.52 g of Top Vision agarose (Thermo Scientific) + 50 ml of TAE 1X.
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| - | <br/>Microwave 30s by 30s until agarose total dissolution
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| - | Gel was cooling down until to be lukewarm, one BET drop was added.
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| - | Gel was loaded with 20 µl of BBa_E0240 PCR product and BBa_I20260 purified PCR product added with 4 µl of loading dye, at 10µl of the mix per well.
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| - | Gel running 45 minutes at 100 mV in TAE 1X buffer.
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| - | <div class="center">
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| - | <div class="thumb tnone">
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| - | <div class="thumbinner" style="width:502px;">
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| - | <a href="URL IMAGE" class="image">
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| - | <img alt="IMAGE" src="https://static.igem.org/mediawiki/2014/a/ae/Gel13082014bis.jpg" width="300px;" class="thumbimage"/>
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| - | </a>
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| - | <div class="thumbcaption">
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| - | <div class="magnify">
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| - | <a href="https://static.igem.org/mediawiki/2014/a/ae/Gel13082014bis.jpg" class="internal" title="Enlarge">
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| - | <img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="Symbol"/>
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| - | </a>
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| - | <center>Figure 2: 1% agarose gel of PCR products from 08.12.2014 after PCR clean up. Lane 4 and 8: Purple 2-Log ladder NEB, Lane 1, 2 and 3: BBa_E0240 purified PCR product, Lane 5, 6 and 7: BBa_I20260 purified PCR product </center>
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| - | <br/>3 pieces of gel were sampled in four tubes to perform a DNA purification from gel. The DNA purification was made with the GeneJET purification kit (Thermo Scientific.
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| - | <br/> A verification electrophoresis was performed on a 1% agarose gel.
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| - | <div class="center">
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| - | <div class="thumbinner" style="width:502px;">
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| - | <a href="URL IMAGE" class="image">
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| - | <img alt="IMAGE" src="https://static.igem.org/mediawiki/2014/a/ae/Gel13082014bis.jpg" width="300px;" class="thumbimage"/>
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| - | </a>
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| - | <div class="thumbcaption">
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| - | <div class="magnify">
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| - | <a href="https://static.igem.org/mediawiki/2014/a/ae/Gel13082014bis.jpg" class="internal" title="Enlarge">
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| - | <img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="Symbol"/>
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| - | </a>
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| - | </div>
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| - | <center>Figure 2: 1% agarose gel of PCR products from 08.12.2014 after PCR clean up. Lane 4 and 8: Purple 2-Log ladder NEB, Lane 1, 2 and 3: BBa_E0240 purified PCR product, Lane 5, 6 and 7: BBa_I20260 purified PCR product </center>
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| - | <h2> 08.15.2014 </h2>
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| - | <h2> 08.16.2014 </h2>
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| - | <h2> 08.17.2014 </h2>
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