Team:UNICAMP-Brazil/Notebooks/September 29

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====Dephosphorylation - CIAP test====
====Dephosphorylation - CIAP test====
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- Only a few colonies were found this morning =(. Unfortunatly the number of colonies wasn´t enough as a parameter to decide between CIAP and SAP. So we decided to use SAP to dephosphorylate our biofusion vector, considering that the protocol is much easier.
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*<p style=”text-align:justify;”>Only a few colonies were found this morning =(. Unfortunately the number of colonies wasn´t enough to compare and decide between CIAP and SAP. So we decided to use SAP to dephosphorylate our biofusion vector, considering that the protocol is much easier.</p>
====New biobricks====
====New biobricks====
-
- Today we performed 5' dephosphorylation of the biofusion vector with SAP (Protocol 9). We did the ligation reaction (Protocol 11) with the 5' dephosphorylated vector and the three digested (XbaI and SpeI) new parts (pJen1, pDLD and Lysozyme).
+
*<p style=”text-align:justify;”>Today we performed 5' dephosphorylation of the biofusion vector with SAP ([https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/SAP_Dephosphorylation Protocol 9]). We did the ligation reaction ([https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/T4_DNA_Ligase Protocol 11]) with the 5' dephosphorylated vector and the three digested (XbaI and SpeI) new parts (pJen1, pDLD and Lysozyme).</p>
-
- We transformed the thermocompetent E. coli (protocol 3) with the ligations and plated in LB+Amp media.
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*<p style=”text-align:justify;”>We transformed the thermocompetent E. coli ([https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/Electroporation Protocol 3]) with the ligations and plated in LB+Amp media.</p>
''Raíssa and Taís''
''Raíssa and Taís''
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==''' ColiGuard '''==
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====Cre-Recombinase's and pSB1A3's purification====
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* Now that we obtained more Cre-Recombinase sample (see September 27th), we could proceed with Cre-Recombinase without ATG's biobrick construction.
 +
* We ran an agarose gel with Cre-Recombinase's PCR product, as well as pSB1A3's miniprep sample. We then purified the target bands with Purelink's Quick Gel Extrection Kit, according to the manufacturer's protocol ([https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/Purification_of_DNA_fragments_from_agarose_gels Protocol 7]).
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''Víctor''
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====CeiB: Colony PCR ====
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*<p style=”text-align:justify;”>Apparently, this time we got transformed cells. We performed colony PCR with VF and VR primers in order to comprove the transformation.  The CeaB transformation was wrong, because it didn’t show the right size band. We tried one more time the transformation. Otherwise, the CeiB transformation showed a possibly correct band size (~400). To confirm it, tomorrow we are going to make miniprep and PCR again.</p>
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[[image:29_set.jpg‎|center]]
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''Luige''
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====Miniprep Results====
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*<p style=”text-align:justify;”>We ran an agarose gel with the minipreps samples (procedure performed yesterday).</p>
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[[image:miniprep2809_17H_17J_24M.jpg]]
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[[image:miniprep2809_12O.jpg|center]]
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*<p style=”text-align:justify;”>According to both pictures, we sucessfully recovered all biobricks! :)</p>
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''Marcelo''
{{:Team:UNICAMP-Brazil/inc_rodape}}
{{:Team:UNICAMP-Brazil/inc_rodape}}

Latest revision as of 03:38, 22 October 2009

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YeastGuard

Dephosphorylation - CIAP test

  • Only a few colonies were found this morning =(. Unfortunately the number of colonies wasn´t enough to compare and decide between CIAP and SAP. So we decided to use SAP to dephosphorylate our biofusion vector, considering that the protocol is much easier.

New biobricks

  • Today we performed 5' dephosphorylation of the biofusion vector with SAP (Protocol 9). We did the ligation reaction (Protocol 11) with the 5' dephosphorylated vector and the three digested (XbaI and SpeI) new parts (pJen1, pDLD and Lysozyme).

  • We transformed the thermocompetent E. coli (Protocol 3) with the ligations and plated in LB+Amp media.


Raíssa and Taís



ColiGuard

Cre-Recombinase's and pSB1A3's purification

  • Now that we obtained more Cre-Recombinase sample (see September 27th), we could proceed with Cre-Recombinase without ATG's biobrick construction.
  • We ran an agarose gel with Cre-Recombinase's PCR product, as well as pSB1A3's miniprep sample. We then purified the target bands with Purelink's Quick Gel Extrection Kit, according to the manufacturer's protocol (Protocol 7).

Víctor

CeiB: Colony PCR

  • Apparently, this time we got transformed cells. We performed colony PCR with VF and VR primers in order to comprove the transformation. The CeaB transformation was wrong, because it didn’t show the right size band. We tried one more time the transformation. Otherwise, the CeiB transformation showed a possibly correct band size (~400). To confirm it, tomorrow we are going to make miniprep and PCR again.

29 set.jpg

Luige

Miniprep Results

  • We ran an agarose gel with the minipreps samples (procedure performed yesterday).

Miniprep2809 17H 17J 24M.jpg

Miniprep2809 12O.jpg


  • According to both pictures, we sucessfully recovered all biobricks! :)


Marcelo




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