Team:UNICAMP-Brazil/Notebooks/September 18

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(New page: ==''' YeastGuard '''== ====Confirming the JEN1 orf, pJEN1, Lisozyme and pDLD biobricks==== * We decided to make a bigger screening of our colonies to find that ones containing the biobri...)
(→New biobricks - Second screening)
 
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{{:Team:UNICAMP-Brazil/inc_topo}}
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{{:Team:UNICAMP-Brazil/inc calendar}}
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__NOTOC__
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==''' ColiGuard '''==
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==== PY Promoter - Miniprep and PCR confirmation ====
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*<p style=”text-align:justify;”>Today we performed minipreps ([https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/Mini-Prep Protocol 2]) to extract the plasmids from the selected colonies inoculated yesterday.</p>
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*<p style=”text-align:justify;”>After the extraction we performed PCR reactions with 3 pairs of primers to confirm the ligation:</p>
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<p style=”text-align:justify;”>- Ppy-F and Ppy-R</p>
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<p style=”text-align:justify;”>- Ppy-F and VR</p>
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<p style=”text-align:justify;”>- VF and VR</p>
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*<p style=”text-align:justify;”>Unfortunately, the sizes of the bands in the agarose gel were not compatible with the expected size of the bands. =(</p>
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''Fabi and Léo''
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==== CeaB, CeiB and BBa_B0015 restriction, purification and quantification ====
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*<p style=”text-align:justify;”>We made the DNA purification in order to clean all buffer PCR salts.  Immediately, we cut CeaB and CeiB purified with ''Spe''I and ''Xba''I restriction enzymes. We made that, because we want to join the CeaB DNA with a terminator. The terminator chosen was BBa_B0015.  Separately, we cut the terminator plasmid with ''Xba''I enzyme restriction and quantificated DNA. Then, we performed DNA purification with Pure-link Purification PCR kit.</p>
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''Luige''
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====Cre-Recombinase and pSB1A3 digestion====
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*<p style=”text-align:justify;”>Today we digested the purified PCR product for Cre-Recombinase without ATG start codon (see Septermber 13th) with ''Xba''I and ''Spe''I restriction enzymes. We also digested the vector pSB1A3 with the same enzymes, aiming in the future ligation between them.</p>
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*<p style=”text-align:justify;”>Digestion lasted 3 hours on both cases.</p>
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''Víctor''
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==''' YeastGuard '''==
==''' YeastGuard '''==
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====Confirming the JEN1 orf, pJEN1, Lisozyme and pDLD biobricks====
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====New biobricks - Second screening====
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*<p style=”text-align:justify;”>This time we tried to find the colonies containing the biobricks by colony PCR (using the forward primer of the insert and the reverse of the plasmid), not digestion. We did 10 reactions of each biobrick. Unfortunately we didn´t find the correct bands.</p>
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[[image:20090918_PCRcol_todos_JEN1orf.JPG.png|350px|center]] 
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''Taís''
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* We decided to make a bigger screening of our colonies to find that ones containing the biobricks. We grew 24 colonies from each biobrick O.N. in liquid media.  
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*<p style=”text-align:justify;”>We decided to make a bigger screening of our colonies to find the ones containing the biobricks (pJen1, pDLD, Lysozyme, ''JEN1'' orf). We grew 24 colonies from each biobrick O.N. in liquid medium.</p>
''Raíssa''
''Raíssa''
{{:Team:UNICAMP-Brazil/inc_rodape}}
{{:Team:UNICAMP-Brazil/inc_rodape}}

Latest revision as of 03:42, 22 October 2009

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ColiGuard

PY Promoter - Miniprep and PCR confirmation

  • Today we performed minipreps (Protocol 2) to extract the plasmids from the selected colonies inoculated yesterday.

  • After the extraction we performed PCR reactions with 3 pairs of primers to confirm the ligation:

- Ppy-F and Ppy-R

- Ppy-F and VR

- VF and VR

  • Unfortunately, the sizes of the bands in the agarose gel were not compatible with the expected size of the bands. =(

Fabi and Léo

CeaB, CeiB and BBa_B0015 restriction, purification and quantification

  • We made the DNA purification in order to clean all buffer PCR salts. Immediately, we cut CeaB and CeiB purified with SpeI and XbaI restriction enzymes. We made that, because we want to join the CeaB DNA with a terminator. The terminator chosen was BBa_B0015. Separately, we cut the terminator plasmid with XbaI enzyme restriction and quantificated DNA. Then, we performed DNA purification with Pure-link Purification PCR kit.

Luige

Cre-Recombinase and pSB1A3 digestion

  • Today we digested the purified PCR product for Cre-Recombinase without ATG start codon (see Septermber 13th) with XbaI and SpeI restriction enzymes. We also digested the vector pSB1A3 with the same enzymes, aiming in the future ligation between them.

  • Digestion lasted 3 hours on both cases.

Víctor

YeastGuard

New biobricks - Second screening

  • This time we tried to find the colonies containing the biobricks by colony PCR (using the forward primer of the insert and the reverse of the plasmid), not digestion. We did 10 reactions of each biobrick. Unfortunately we didn´t find the correct bands.

20090918 PCRcol todos JEN1orf.JPG.png


Taís

  • We decided to make a bigger screening of our colonies to find the ones containing the biobricks (pJen1, pDLD, Lysozyme, JEN1 orf). We grew 24 colonies from each biobrick O.N. in liquid medium.

Raíssa




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