Team:UNITN-Trento/Notebook/Labposts/07/19

From 2013.igem.org

(Difference between revisions)
 
(One intermediate revision not shown)
Line 1: Line 1:
-
{
+
{"date":"2013-07-06","author":"viola","title":"<html>cloning of EFE in pSpac and pXyl vectors</html>","content":"<html> Today i start the cloning to insert the ethylene forming enzyme in both the bacillus plasmids. To make this i digested the vectors and also EFE+B0015 in pSB1C3 with EcorI and PstI. {{:Team:UNITN-Trento/Templates/Styles/Spoiler|LIGATION TABLE 1| <html> <center> <table> <tr> <th>LIGATION</th> <th>EFE+B0015 in pSPAC</th> <th>EFE+B0015 in pXYL</th> </tr> <tr> <th>PLASMID CONC.</th> <td>20 µl</td> <td>20 µl</td> </tr> <tr> <th>PLASMID LENGHT</th> <td>7082 BP</td> <td>3594 BP</td> </tr> <tr> <th>INSERT CONCENTRATION</th> <td>20 µl</td> <td>20 µl</td> </tr> <tr> <th>INSERT LENGHT</th> <td>1251 BP</td> <td>1251 BP</td> </tr> <tr> <th>PLASMID USED</th> <td>50 ng/µl</td> <td>50 ng/µl</td> </tr> <tr> <th>REACTION VOLUME</th> <td>20 µl</td> <td>20 µl</td> </tr> <tr> <th>BUFFER</th> <td>10 µl</td> <td>10 µl</td> </tr> </table> </center> </html>}} <html> </html>}} {{:Team:UNITN-Trento/Templates/Styles/Spoiler|LIGATION TABLE PSPAC| <html> <center> <table> <tr> <th><font color=\"red\">EFE IN PSPAC</font></th> <th>CTRL</th> <th>1:1</th> <th>1:2</th> <th>1:4</th> </tr> <tr> <th>INSERT (µl)</th> <td>0</td> <td>0.44</td> <td>0.88</td> <td>1.76</td> </tr> <tr> <th>WATER (µl)</th> <td>14.5 </td> <td>14.06</td> <td>13.62</td> <td>12.74</td> </tr> <tr> <th>LIGASE (µl)</th> <td>1 </td> <td>1 </td> <td>1</td> <td>1</td> </tr> <tr> <th>PLASMID (µl)</th> <td>2.5</td> <td>2.5</td> <td>2.5</td> <td>2.5</td> </tr> <tr> <th>BUFFER (µl)</th> <td>2</td> <td>2</td> <td>2</td> <td>2</td> </tr> </table> </center> </html>}} {{:Team:UNITN-Trento/Templates/Styles/Spoiler|LIGATION TABLE PXYL| <html> <center> <table> <tr> <th><font color=\"red\">EFE IN PXYL</font></th> <th>CTRL</th> <th>1:1</th> <th>1:2</th> <th>1:4</th> </tr> <tr> <th>INSERT (µl)</th> <td>0</td> <td>0.87</td> <td>1.74</td> <td>3.48</td> </tr> <tr> <th>WATER (µl)</th> <td>14.5 </td> <td>13.63</td> <td>12.76</td> <td>11.02</td> </tr> <tr> <th>LIGASE (µl)</th> <td>1 </td> <td>1 </td> <td>1</td> <td>1</td> </tr> <tr> <th>PLASMID (µl)</th> <td>2.5</td> <td>2.5</td> <td>2.5</td> <td>2.5</td> </tr> <tr> <th>BUFFER (µl)</th> <td>2</td> <td>2</td> <td>2</td> <td>2</td> </tr> </table> </center> </html>}} <html> </html>}} <html> </html>}} </html>","tags":"BACILLUS"}
-
"date" : "2013-07-22",
+
-
"author" : "fabio",
+
-
"title" : " blue and red light sensors!!",
+
-
"content" : "<html> let’s get ready to screen!! This time I amplified my parts by pcr them out with OneTaq polymerase!! I started from the blue promoter (number A from the firt  set). Pcr sample composition: <br>5X Onetaq standard reaction buffer  10 ul <br>10 mM dNTPs  1 ul<br>Forward primer 1ul<br>Reverse primer 1 ul<br>One Taq 0.25<br>Phusion 0.3<br>Template 0.5<br>Water up to 50 ul<br>Pcr program values: <br>Initial digestion: 94° 2min <br>Digestion: 94 30 sec<br>Annealing: 60 1 min<br>Annealing: 68° 15 sec<br>Final extention: 68° 5 min<br>(30 cycles from step 3 to step 5) <br>The screening was successful : <br> </html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|gel|<html><center><img src=\"https://static.igem.org/mediawiki/2013/0/06/Tn-2013_C.jpg\" width=\"450px\" /></center></html>}}<html>Finally I purified the PCR and abtained a 209,8 ng/ul yieald.Meanwhile I started thinking about the ligation of the two blue parts: the idea is to use k592016 as plasmid and k592006 as an insert to put downstream . So I had to digest o/n  3 ug of 016(from the first set) with Spel and Pstl, and 006( the purified Pcr) with Xbal and Pstl. At the same time I digested R0010 with Ecorl and Spel, cause I’ll need to put it on top of everything once I have my biobrick.</html>",
+
-
"tags" : " YF1_FixJ - FixK2- ho1_PcyA_cph8-OmpR"
+
-
}
+

Latest revision as of 10:36, 3 October 2013

{"date":"2013-07-06","author":"viola","title":"cloning of EFE in pSpac and pXyl vectors","content":" Today i start the cloning to insert the ethylene forming enzyme in both the bacillus plasmids. To make this i digested the vectors and also EFE+B0015 in pSB1C3 with EcorI and PstI. {{:Team:UNITN-Trento/Templates/Styles/Spoiler|LIGATION TABLE 1|

LIGATION EFE+B0015 in pSPAC EFE+B0015 in pXYL
PLASMID CONC. 20 µl 20 µl
PLASMID LENGHT 7082 BP 3594 BP
INSERT CONCENTRATION 20 µl 20 µl
INSERT LENGHT 1251 BP 1251 BP
PLASMID USED 50 ng/µl 50 ng/µl
REACTION VOLUME 20 µl 20 µl
BUFFER 10 µl 10 µl
}} }}
LIGATION TABLE PSPAC
EFE IN PSPAC CTRL 1:1 1:2 1:4
INSERT (µl) 0 0.44 0.88 1.76
WATER (µl) 14.5 14.06 13.62 12.74
LIGASE (µl) 1 1 1 1
PLASMID (µl) 2.5 2.5 2.5 2.5
BUFFER (µl) 2 2 2 2
LIGATION TABLE PXYL
EFE IN PXYL CTRL 1:1 1:2 1:4
INSERT (µl) 0 0.87 1.74 3.48
WATER (µl) 14.5 13.63 12.76 11.02
LIGASE (µl) 1 1 1 1
PLASMID (µl) 2.5 2.5 2.5 2.5
BUFFER (µl) 2 2 2 2
}} }} </html>","tags":"BACILLUS"}

Retrieved from "http://2013.igem.org/Team:UNITN-Trento/Notebook/Labposts/07/19"