Team:UNICAMP-Brazil/Notebooks/September 28

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''Marcelo''
''Marcelo''
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==== PY Promoter - Digestion and ligation reactions ====
 
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*<p style=”text-align:justify;”>Today we digested PY1 + pGEM with EcoRI and SpeI to excise PY fragment from pGEM vector.</p>
 
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*<p style=”text-align:justify;”>We also digested the plasmid BBa_J23100 (that one with the RFP reporter) with EcoRI and SpeI. With this plasmid digested with this 2 enzymes it is going to be possible to insert our digested PY fragment into it.</p>
 
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*<p style=”text-align:justify;”>This digestion reactions lasted for 3 hours.</p>
 
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*<p style=”text-align:justify;”>After the digestion we performed the ligation reaction of PY1 + BBa_J23100 following [https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/T4_DNA_Ligase Protocol 11].</p>
 
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''Fabi and Léo''
 
==== CeaB and CeiB transformation into plasmid [http://partsregistry.org/Part:pSB1A3 pSBA3] ====
==== CeaB and CeiB transformation into plasmid [http://partsregistry.org/Part:pSB1A3 pSBA3] ====
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*<p style=”text-align:justify;”>Today, we dephosphorilated the plasmid [http://partsregistry.org/Part:pSB1A3 pSBA3], immediately started the ligation reaction of this unphosphorylated plasmid with CeaB and CeiB respectively. In view of for this dephosphorylation use buffer salt that could be interfering in the next transformation, we performed a dyalisis so that get samples free salts. At last, we transformated competent E. coli (according to the [https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/Electroporation protocol 3]) and plated the respective plates. This time we expect to obtain transforming cells. .</p>
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*<p style=”text-align:justify;”>Today, we dephosphorilated the plasmid [http://partsregistry.org/Part:pSB1A3 pSBA3], and immediately started the ligation reaction of this unphosphorylated plasmid with CeaB and CeiB. Considering that the dephosphorylation use buffer that contains salt and that salt could be interfering in the transformations, we performed a dyalisis to get the samples free of salts. At last, we transformed competent ''E. coli'' (according to the [https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/Electroporation protocol 3]) and plated the respective plates. This time we expect to obtain transforming cells.</p>
''Luige''
''Luige''
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==''' YeastGuard '''==
==''' YeastGuard '''==

Latest revision as of 03:26, 22 October 2009

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ColiGuard

Minipreps from BBa_E0840, BBa_I718017, BBa_J61000 and BBa_I78016

  • Today we performed minipreps from yesterday's inoculated cultures, related to the biobricks already cited.

  • We followed Protocol 2, without modification.

Marcelo


Purification of pTet and double terminator biobricks

  • Once we digested those biobricks yesterday, we could proceed today to the purification of the target band from an agarose gel.

  • We used Invitrogen's Purelink Quick Gel Extraction Kit, following the manufacturer's protocol (Protocol 7), without modifications.

Marcelo


CeaB and CeiB transformation into plasmid [http://partsregistry.org/Part:pSB1A3 pSBA3]

  • Today, we dephosphorilated the plasmid [http://partsregistry.org/Part:pSB1A3 pSBA3], and immediately started the ligation reaction of this unphosphorylated plasmid with CeaB and CeiB. Considering that the dephosphorylation use buffer that contains salt and that salt could be interfering in the transformations, we performed a dyalisis to get the samples free of salts. At last, we transformed competent E. coli (according to the protocol 3) and plated the respective plates. This time we expect to obtain transforming cells.

Luige

YeastGuard

Dephosphorylation - CIAP test

  • The E.coli didn´t grow in any of the plates again. =[ We discovered that the electrocompetent E.coli that we used yesterday were dead! So we transformed another electrocompetent E.coli using the rest of the ligation reactions we had. We extremely hope to find colonies tomorrow!

Raíssa and Taís




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