Team:Marburg:Project:Notebook:May
From 2014.igem.org
(Difference between revisions)
Line 342: | Line 342: | ||
</tr> | </tr> | ||
</table> | </table> | ||
- | <p>The isolated plasmids were | + | <p>The isolated plasmids were digested with <i>Nco</i>I for 1h 20 min and further analyzed on a 1% agarose gel.</p> |
</div> | </div> | ||
<div class="results"> | <div class="results"> | ||
<h3>Results</h3> | <h3>Results</h3> | ||
<img src="https://static.igem.org/mediawiki/2014/5/5e/MR_2014-05-06_NcoI.PNG" width="30%" /> | <img src="https://static.igem.org/mediawiki/2014/5/5e/MR_2014-05-06_NcoI.PNG" width="30%" /> | ||
- | <p>Restriction of the isolated pMa12 with <i>Nco</i>I; the lanes contain the | + | <p>Restriction of the isolated pMa12 with <i>Nco</i>I; the lanes contain the digested pMa12 in the order of the picked clones 1 - 15; clone 9 was negative; the lanes between the restrictions and the marker contain the isolated pMAD but it appears to be empty.<br /> |
The most clones contained a plasmid that has only one <i>Nco</i>I restriction site. The expected size of the pMa12 should be around 6.6 kb. All fragments are of the expected size.</p> | The most clones contained a plasmid that has only one <i>Nco</i>I restriction site. The expected size of the pMa12 should be around 6.6 kb. All fragments are of the expected size.</p> | ||
</div> | </div> | ||
Line 502: | Line 502: | ||
</fieldset> | </fieldset> | ||
<fieldset class="exp13"> | <fieldset class="exp13"> | ||
- | <legend><a name="exp13.43">13.43 Ligation of the annealed oligos into | + | <legend><a name="exp13.43">13.43 Ligation of the annealed oligos into digested piGEM001</a></legend> |
<div class="aim"> | <div class="aim"> | ||
<p>Aim: Create plasmid with the corresponding promoter sequences for Ag/Cu</p> | <p>Aim: Create plasmid with the corresponding promoter sequences for Ag/Cu</p> | ||
Line 1,310: | Line 1,310: | ||
</div> | </div> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
- | <p>The plasmid pMAD was isolated and | + | <p>The plasmid pMAD was isolated and digested with the restriction enzymes <i>Bam</i>HI and <i>Nco</i>I to create fitting ends for the ligation with the digested PCR-fragment.</p> |
<table width="100%" border="1"> | <table width="100%" border="1"> | ||
<tr> | <tr> | ||
Line 1,345: | Line 1,345: | ||
</fieldset> | </fieldset> | ||
<fieldset class="exp15"> | <fieldset class="exp15"> | ||
- | <legend><a name="exp15.34">15.34 Ligation of | + | <legend><a name="exp15.34">15.34 Ligation of digested pMAD with the digested hag/flank-Construct and trafo</a></legend> |
<div class="aim"> | <div class="aim"> | ||
<p>Aim: link the fragment with the vector and trafo of DH5α</p> | <p>Aim: link the fragment with the vector and trafo of DH5α</p> | ||
</div> | </div> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
- | <p>The ligation of pMAD and the | + | <p>The ligation of pMAD and the digested PCR-fragment was done according to the previous mentioned ligation calculator.</p> |
<table width="100%" border="1"> | <table width="100%" border="1"> | ||
<tr> | <tr> | ||
Line 1,463: | Line 1,463: | ||
</div> | </div> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
- | <p>The plasmid pMAD from 13.05.14 (ca. 25µl) and | + | <p>The plasmid pMAD from 13.05.14 (ca. 25µl) and digested with the restriction enzymes <i>Bam</i>HI and <i>Nco</i>I to create fitting ends for the ligation with the digested PCR-fragment. The digest was performed in 2 Eppis with 12,5µl plasmid each.</p> |
<table width="100%" border="1"> | <table width="100%" border="1"> | ||
<tr> | <tr> | ||
Line 1,542: | Line 1,542: | ||
</div> | </div> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
- | <p>The ligation of pMAD and the | + | <p>The ligation of pMAD and the digested PCR-fragment was done according to the previous ligation calculator.</p> |
<table width="100%" border="1"> | <table width="100%" border="1"> | ||
<tr> | <tr> | ||
Line 2,232: | Line 2,232: | ||
<fieldset class="exp15"> | <fieldset class="exp15"> | ||
- | <legend><a name="exp15.45">15.45 Gibson Assembly of | + | <legend><a name="exp15.45">15.45 Gibson Assembly of digested pMad-fla and cup 1 and Transformation into <em>E.Coli</em> XL1-Blue</a></legend> |
<div class="aim"> | <div class="aim"> | ||
<p>Aim: create a flagellin that contains the metallothinein cup1</p> | <p>Aim: create a flagellin that contains the metallothinein cup1</p> | ||
Line 2,445: | Line 2,445: | ||
</table> | </table> | ||
<p>The restriction was carried out at 37°C for 1h.<br /> | <p>The restriction was carried out at 37°C for 1h.<br /> | ||
- | The restriction was cleaned with a DNA Gel Ex Kit (c = 40 ng/µl). 4µl (160 ng) of this | + | The restriction was cleaned with a DNA Gel Ex Kit (c = 40 ng/µl). 4µl (160 ng) of this digested plasmid were mixed with 1 µl of the cup1-1 fragment (9.6 ng/µl). These 5 µl were inserted into the prepared 15 µl Gibson-mix. The reaction was incubated at RT for 1 min and then at 50°C for 1 hour. An extra Gibson-reaction was carried out as a control with just the linearized pMAD without the <i>cup</i>1-1.<br /> |
<em>E. coli</em> DH5α cells were transformed with the Gibson reactions and plated out on LB-Amp. They were incubated at 37°C over night.</p> | <em>E. coli</em> DH5α cells were transformed with the Gibson reactions and plated out on LB-Amp. They were incubated at 37°C over night.</p> | ||
</div> | </div> | ||
Line 2,591: | Line 2,591: | ||
</table> | </table> | ||
<p>The restriction was incubated at 37°C for 1 h.</p> | <p>The restriction was incubated at 37°C for 1 h.</p> | ||
- | <p>The restriction mix was purified with the Gel Ex Kit. The yield was too low (c = 3.1 ng/µl) to use it for a Gibson-Assembly, so the rest of the pET24d-fla was | + | <p>The restriction mix was purified with the Gel Ex Kit. The yield was too low (c = 3.1 ng/µl) to use it for a Gibson-Assembly, so the rest of the pET24d-fla was digested with <i>Spe</i>I.</p> |
<table width="100%" border="1"> | <table width="100%" border="1"> | ||
<tr> | <tr> | ||
Line 2,619: | Line 2,619: | ||
</table> | </table> | ||
<p>The restriction was incubated at 37°C for 1h.</p> | <p>The restriction was incubated at 37°C for 1h.</p> | ||
- | <p>The restriction mix was purified with the Gel Ex Kit (c = 20.1 ng/µl), which was enough for the Gibson-Assembly. 4µl (80 ng) of this | + | <p>The restriction mix was purified with the Gel Ex Kit (c = 20.1 ng/µl), which was enough for the Gibson-Assembly. 4µl (80 ng) of this digested plasmid were mixed with 1 µl of the cup1-1 fragment (9.6 ng/µl). These 5 µl were inserted into the prepared 15µl Gibson-mix. The reaction was incubated at RT for 1 min and then at 50°C for 1 h. An extra Gibson-reaction was carried out as a control with just the linearized pET24d-fla without the <i>cup</i>1-1.</p> |
<p>Competent <em>E. coli</em> XLI-Blue were transformed with the whole Gibson-reactions. They were plated on LB-Kan an incubated at 37°C overnight.</p> | <p>Competent <em>E. coli</em> XLI-Blue were transformed with the whole Gibson-reactions. They were plated on LB-Kan an incubated at 37°C overnight.</p> | ||
</div> | </div> | ||
Line 3,387: | Line 3,387: | ||
<div class="exp-content"> | <div class="exp-content"> | ||
<p>The positive clone 3 from the colony PCR (28.05.14) was used to inoculate 6 ml LB-Amp for a miniprep in order to receive the plasmid for transforming <em>Bacillus subtilis</em>.<br /> | <p>The positive clone 3 from the colony PCR (28.05.14) was used to inoculate 6 ml LB-Amp for a miniprep in order to receive the plasmid for transforming <em>Bacillus subtilis</em>.<br /> | ||
- | Incubation at room temperature over | + | Incubation at room temperature over two days.</p> |
</div> | </div> | ||
</fieldset> | </fieldset> |
Revision as of 13:08, 16 October 2014
Notebook: May