"
Team:UPO-Sevilla/Notebook/07 28
From 2010.igem.org
(New page: <div class=globalBC> {{:Team:UPO-Sevilla/header}} <!-- --> <html> <head> <script type="text/javascript" language="javascript"> <!-- current("notebook","http...) |
|||
| (2 intermediate revisions not shown) | |||
| Line 21: | Line 21: | ||
<p><strong>Paola Gallardo.</strong> We run an electrophoresis to check the result and we observed a lot of unspecific very intense bands, but the expected bands had few intensity. We isolated and purified the desired bands. We digested the products: <i>gltD</i> with XbaI and PstI, <i>fecI-fecR</i> with EcoRI and PstI for two hours. We joined them with different vectors and rested o/n.</p> | <p><strong>Paola Gallardo.</strong> We run an electrophoresis to check the result and we observed a lot of unspecific very intense bands, but the expected bands had few intensity. We isolated and purified the desired bands. We digested the products: <i>gltD</i> with XbaI and PstI, <i>fecI-fecR</i> with EcoRI and PstI for two hours. We joined them with different vectors and rested o/n.</p> | ||
| - | <p><strong>David Caballero.</strong> <i>fecA*</i> inocula appeared in red color behind being incubating all night long. This meant that bacteria had plasmids with RFP (red fluorescent protein) instead of <i>fecA*</i>. So we could not use it. Checking <i>fecA*</i> plates we saw that colonies which come from our inocula | + | <p><strong>David Caballero.</strong> <i>fecA*</i> inocula appeared in red color behind being incubating all night long. This meant that bacteria had plasmids with RFP (red fluorescent protein) instead of <i>fecA*</i>. So we could not use it. Checking <i>fecA*</i> plates we saw that colonies which come from our inocula were red two days after starting the incubation. We found a new white colony too, so we analyzed it by colony PCR and electrophoresis analysis, but the result was negative. Changing focus, we analyzed <i>gltB</i> and <i>fecI-fecR*</i> inocula: vector purification, restriction enzyme parts digestion and 0.8% agarose gel electrophoresis analysis. We had a positive result for <i>gltB</i> part and negative for <i>fecI-fecR*</i> part (not well digested). With this it had been tested a new synthesized part: <i>gltB.</i> The loss of <i>fecI-fecR*</i> is unimportant, since it had already been gained in another process made by a partner. By this time, we had got all searched parts by PCR saving <i>fecA*</i>. It was resisting.</p> |
<h2>Assembly Team</h2> | <h2>Assembly Team</h2> | ||
| Line 28: | Line 28: | ||
<p>And again, we start with UPO 1+19 and UPO 12+2, 12+19, 13+3 y 1+2+16+3.</p> | <p>And again, we start with UPO 1+19 and UPO 12+2, 12+19, 13+3 y 1+2+16+3.</p> | ||
| + | |||
| + | |||
| + | <a class="return_button" href="/Team:UPO-Sevilla/Notebook/07_27" title="Go to 27th of July"><span>Go to 27th of July</span></a> | ||
| + | |||
| + | <a class="next_button" href="/Team:UPO-Sevilla/Notebook/07_29" title="Go to 29th of July"><span>Go to 29th of July</span></a> | ||
| + | |||
| + | <div class="clear"></div> | ||
<a class="return_button" href="/Team:UPO-Sevilla/Notebook" title="Notebook"><span>Return to Notebook</span></a> | <a class="return_button" href="/Team:UPO-Sevilla/Notebook" title="Notebook"><span>Return to Notebook</span></a> | ||
Latest revision as of 21:59, 26 October 2010
July, 28th
Production Team
Paola Gallardo. We run an electrophoresis to check the result and we observed a lot of unspecific very intense bands, but the expected bands had few intensity. We isolated and purified the desired bands. We digested the products: gltD with XbaI and PstI, fecI-fecR with EcoRI and PstI for two hours. We joined them with different vectors and rested o/n.
David Caballero. fecA* inocula appeared in red color behind being incubating all night long. This meant that bacteria had plasmids with RFP (red fluorescent protein) instead of fecA*. So we could not use it. Checking fecA* plates we saw that colonies which come from our inocula were red two days after starting the incubation. We found a new white colony too, so we analyzed it by colony PCR and electrophoresis analysis, but the result was negative. Changing focus, we analyzed gltB and fecI-fecR* inocula: vector purification, restriction enzyme parts digestion and 0.8% agarose gel electrophoresis analysis. We had a positive result for gltB part and negative for fecI-fecR* part (not well digested). With this it had been tested a new synthesized part: gltB. The loss of fecI-fecR* is unimportant, since it had already been gained in another process made by a partner. By this time, we had got all searched parts by PCR saving fecA*. It was resisting.
Assembly Team
We have to test the UPO 1+2 candidate so we digest with Nhe1 to UPO1 and we can see the plasmid run into the gel like a lineal plasmid. Thus, finally we can say we have UPO 1+2.
And again, we start with UPO 1+19 and UPO 12+2, 12+19, 13+3 y 1+2+16+3.
Go to 27th of July Go to 29th of July Return to Notebook
Sponsors
