Team:UNICAMP-Brazil/Notebooks/September 30

From 2009.igem.org

(Difference between revisions)
(→New biobricks - screening)
(→Customizing the PCR)
 
(25 intermediate revisions not shown)
Line 6: Line 6:
==''' YeastGuard '''==
==''' YeastGuard '''==
-
====New biobricks - screening====
+
====New biobricks - still screening====
-
- This morning we found some ''E. coli'' colonies that were transformed yesterday! We performed PCR with 10 colonies from each biobrick (pJEN1, pDLD and Lysozyme) with the vector's primers (VR and VF).   
+
*<p style=”text-align:justify;”>This morning we found some ''E. coli'' colonies that were transformed yesterday! We performed PCR with 10 colonies from each biobrick (pJEN1, pDLD and Lysozyme) with the vector's primers (VR and VF).</p>  
-
- The figure shows that unfortunately none of the colonies tested had the insert into the vector. The size of the amplicons correspond to the vector's fragment without any insert (+/- 1400bp).  
+
*<p style=”text-align:justify;”>The figure shows that unfortunately none of the colonies tested had the insert into the vector even with its dephosphorylation. =( The size of the amplicons correspond to the vector's fragment without any insert (+/- 1400bp).</p>
-
GEL
+
[[Image:UNICAMP_20090930_GEL1.png|500px|center]]
-
- We also decided to try to do a miniprep using the rest of the transformed ''E. coli'' that was not plated in LB+AMP media. So today we made the inoculum.
+
*<p style=”text-align:justify;”>We also decided to try to do a miniprep using the rest of the transformed ''E. coli'' that was not plated in LB+AMP media. So today we made the inoculum.</p>
 +
 
 +
*<p style=”text-align:justify;”>Thinking about another strategy to avoid recircularization and antisense insertion...</p>
''Raíssa and Taís''
''Raíssa and Taís''
 +
====Customizing the PCR====
 +
*<p style=”text-align:justify;”>Electrophoresis of the Jen1 (ORF) product PCR to purify the band and proceed a new ''JEN1'' (ORF) PCR.</p> 
 +
 +
[[Image:Jen1.JPG‎|center]]
 +
 +
 +
 +
''Wesley and Gleidson''
 +
 +
==''' ColiGuard '''==
 +
 +
====Digestion of Biobricks BBa_E0840, BBa_I718017, BBa_J61000 and BBa_I718016====
 +
 +
* After confirming the miniprep procedure, we digested the cited biobricks with the correct enzymes in order to make possible the construction of our necessary devices. Therefore, we had to:
 +
 +
- BBa_E0840: isolate the part in order to ligate it in the downstream position of another biobrick (digested with XpeI and PstI)
 +
 +
- BBa_I718017: open the biobrick in the downstream position of it's part (digested with SpeI and PstI)
 +
 +
- BBa_J61000: isolate the part in order to insert it in the upstream position of another biobrick (digested with EcoRI and SpeI)
 +
 +
- BBa_I718016: open the biobrick in the upstream position of it's part (digested with EcoRI and XbaI)
 +
 +
 +
* Digestion lasted 3 hours on all cases.
 +
 +
 +
''Marcelo''
 +
 +
====Digestion of Biobricks BBa_E0840, BBa_I718017, BBa_J61000 and BBa_I718016====
 +
 +
*<p style=”text-align:justify;”>Today, we performed miniprep ([https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/Mini-Prep Protocol 2]) of the selected colonies yesterday. After that, we performed two PCR. The first reaction with VF2 and VR primers and the second reaction with Anticol F and Anticol R primers (designed by us) in order to comprove the correct transformation. Lamentably, we didn’t get it. In addition we performed a digestion reaction with ''EcoR''I and ''Pst''I.  We didn’t see the expected band size.  Definitively, the CeiB isn’t into the  plasmid. We tried a search. Otherwise, we repeated the cell transformation with CeaB DNA and colony PCR but didn’t see the right size band.</p>
 +
 +
''Luige''
{{:Team:UNICAMP-Brazil/inc_rodape}}
{{:Team:UNICAMP-Brazil/inc_rodape}}

Latest revision as of 03:47, 22 October 2009

Topo l2.gif topo_r_igem.gif
topo_r_b.gif
Back to Calendar

YeastGuard

New biobricks - still screening

  • This morning we found some E. coli colonies that were transformed yesterday! We performed PCR with 10 colonies from each biobrick (pJEN1, pDLD and Lysozyme) with the vector's primers (VR and VF).

  • The figure shows that unfortunately none of the colonies tested had the insert into the vector even with its dephosphorylation. =( The size of the amplicons correspond to the vector's fragment without any insert (+/- 1400bp).

UNICAMP 20090930 GEL1.png

  • We also decided to try to do a miniprep using the rest of the transformed E. coli that was not plated in LB+AMP media. So today we made the inoculum.

  • Thinking about another strategy to avoid recircularization and antisense insertion...

Raíssa and Taís

Customizing the PCR

  • Electrophoresis of the Jen1 (ORF) product PCR to purify the band and proceed a new JEN1 (ORF) PCR.

Jen1.JPG


Wesley and Gleidson

ColiGuard

Digestion of Biobricks BBa_E0840, BBa_I718017, BBa_J61000 and BBa_I718016

  • After confirming the miniprep procedure, we digested the cited biobricks with the correct enzymes in order to make possible the construction of our necessary devices. Therefore, we had to:

- BBa_E0840: isolate the part in order to ligate it in the downstream position of another biobrick (digested with XpeI and PstI)

- BBa_I718017: open the biobrick in the downstream position of it's part (digested with SpeI and PstI)

- BBa_J61000: isolate the part in order to insert it in the upstream position of another biobrick (digested with EcoRI and SpeI)

- BBa_I718016: open the biobrick in the upstream position of it's part (digested with EcoRI and XbaI)


  • Digestion lasted 3 hours on all cases.


Marcelo

Digestion of Biobricks BBa_E0840, BBa_I718017, BBa_J61000 and BBa_I718016

  • Today, we performed miniprep (Protocol 2) of the selected colonies yesterday. After that, we performed two PCR. The first reaction with VF2 and VR primers and the second reaction with Anticol F and Anticol R primers (designed by us) in order to comprove the correct transformation. Lamentably, we didn’t get it. In addition we performed a digestion reaction with EcoRI and PstI. We didn’t see the expected band size. Definitively, the CeiB isn’t into the plasmid. We tried a search. Otherwise, we repeated the cell transformation with CeaB DNA and colony PCR but didn’t see the right size band.

Luige




Retrieved from "http://2009.igem.org/Team:UNICAMP-Brazil/Notebooks/September_30"