Team:UNICAMP-Brazil/Notebooks/October 11

From 2009.igem.org

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====PCR colony of the BBa B0014 + BBa K112806 Ligation====
====PCR colony of the BBa B0014 + BBa K112806 Ligation====
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*<p style=”text-align:justify;”>We chose 9 colonies to do the PCR, 3 results were positive, the colonies 2, 5 and 7 and appeared in the agarose gel with the expected size! 2 colonies was used to do a innoculum that will be used in a miniprep tomorrow </p>
+
*<p style=”text-align:justify;”>We chose 9 colonies to do the PCR, 3 results were positive, the colonies 2, 5 and 7 and appeared in the agarose gel with the expected size! 2 colonies was used to do a innoculum that will be used in a miniprep tomorrow.</p>
[[Image:PCRB0014 K112806..JPG|center]]
[[Image:PCRB0014 K112806..JPG|center]]
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====finOP-pGEM digestion purification====
====finOP-pGEM digestion purification====
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* After confirming correctly finOP with pGEM ligations, today we gather all confirmed samples (for finO and for finP) into a single sample for each one.
+
*<p style=”text-align:justify;”>After confirming correctly finOP with pGEM ligations, today we gather all confirmed samples (for finO and for finP) into a single sample for each one.</p>
-
* We ran 40 uL of each gathered sample in an agarose gel for later purification. Unfortunately, finP band appeared extremely weak in the gel and we weren't able to purify it. As for finO, it's band appeared in gel, but we decided to wait for finP in order to perform both purifications together.
+
*<p style=”text-align:justify;”>We ran 40 uL of each gathered sample in an agarose gel for later purification. Unfortunately, finP band appeared extremely weak in the gel and we weren't able to purify it. As for finO, it's band appeared in gel, but we decided to wait for finP in order to perform both purifications together.</p>
-
* Thus, we took all the gathered finP sample left and concentrated it in speed vacuum, until ir reaches about 20 uL. Then we ran another agarose gel with this concentrated sample and with another 40 uL from finO gathered sample.
+
*<p style=”text-align:justify;”>Thus, we took all the gathered finP sample left and concentrated it in speed vacuum, until ir reaches about 20 uL. Then we ran another agarose gel with this concentrated sample and with another 40 uL from finO gathered sample.</p>
-
* This time, finP appeared as a barely visible band, but we were able to purify it, as we did for finO to.
+
*<p style=”text-align:justify;”>This time, finP appeared as a barely visible band, but we were able to purify it, as we did for finO to.</p>
-
* We performed purification using Invitrogen's Purelink Quick Gel Extractin Kit, following manufacturer's protocol ([https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/Purification_of_DNA_fragments_from_agarose_gels Protocol 7]) without modifications).
+
*<p style=”text-align:justify;”>We performed purification using Invitrogen's Purelink Quick Gel Extractin Kit, following manufacturer's protocol ([https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/Purification_of_DNA_fragments_from_agarose_gels Protocol 7]) without modifications).</p>
''Marcelo''
''Marcelo''
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====finOP purification results====
====finOP purification results====
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* After purification procedure, we quantified total DNA present in both samples.
+
*<p style=”text-align:justify;”>After purification procedure, we quantified total DNA present in both samples.</p>
-
* finO resulted in 15 ng/uL. Although very low, this amount might be enough for proceeding in biobrick construction.
+
*<p style=”text-align:justify;”>finO resulted in 15 ng/uL. Although very low, this amount might be enough for proceeding in biobrick construction.</p>
-
* As for finP, no amount could be measured. =/ Probably, the barely visible band hadn't enough DNA for detection or there was a significant loss during purification process.
+
*<p style=”text-align:justify;”>As for finP, no amount could be measured. =/ Probably, the barely visible band hadn't enough DNA for detection or there was a significant loss during purification process.</p>
''Marcelo''
''Marcelo''
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====finO====
====finO====
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* Even with no longer finP available, we continued finO's work by ligating finO purified sample into biofusion digested vector, according to [https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/T4_DNA_Ligase Protocol 11].
+
*<p style=”text-align:justify;”>Even with no longer finP available, we continued finO's work by ligating finO purified sample into biofusion digested vector, according to [https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/T4_DNA_Ligase Protocol 11].</p>
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* We then dialyzed the ligation product for 20 minutes and transformed them into electrocompetent ''E. coli'' bacteria, strain DH10B, according to [https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/Electroporation Protocol 3].
+
*<p style=”text-align:justify;”>We then dialyzed the ligation product for 20 minutes and transformed them into electrocompetent ''E. coli'' bacteria, strain DH10B, according to [https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/Electroporation Protocol 3].</p>
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* After one hour being incubated at 37 ºC, transformed cells were plated into LB-AMP media.
+
*<p style=”text-align:justify;”>After one hour being incubated at 37 ºC, transformed cells were plated into LB-AMP media.</p>
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* Plates were incubated at 37 ºC for an O/N period.
+
*<p style=”text-align:justify;”>Plates were incubated at 37 ºC for an O/N period.</p>
''Marcelo''
''Marcelo''
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==''' YeastGuard '''==
==''' YeastGuard '''==
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====pADH1+YFP (new biobrick)====
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====pADH1+YFP====
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* The colonies grew up very well in the plates. So, we selected ten colonies to grow in liquid ?meio? O/N.
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*<p style=”text-align:justify;”>The colonies grew up very well in the plates. So, we selected 10 colonies to grow in liquid LB+AMP media O/N.</p>
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====YEP vector====
====YEP vector====
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* Purification of the bands that correspond to the YEP size digested with PvuII. Then we recirculamos the YEP trough a ligation reaction.
+
*<p style=”text-align:justify;”>Purification of the bands that correspond to the YEP size digested with PvuII. Then we recirculamos the YEP trough a ligation reaction.</p>
[[Image:Yep.JPG|center]]
[[Image:Yep.JPG|center]]

Revision as of 02:08, 21 October 2009

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ColiGuard

PCR colony of the BBa B0014 + BBa K112806 Ligation

  • We chose 9 colonies to do the PCR, 3 results were positive, the colonies 2, 5 and 7 and appeared in the agarose gel with the expected size! 2 colonies was used to do a innoculum that will be used in a miniprep tomorrow.

PCRB0014 K112806..JPG

Luige

finOP-pGEM digestion purification

  • After confirming correctly finOP with pGEM ligations, today we gather all confirmed samples (for finO and for finP) into a single sample for each one.

  • We ran 40 uL of each gathered sample in an agarose gel for later purification. Unfortunately, finP band appeared extremely weak in the gel and we weren't able to purify it. As for finO, it's band appeared in gel, but we decided to wait for finP in order to perform both purifications together.

  • Thus, we took all the gathered finP sample left and concentrated it in speed vacuum, until ir reaches about 20 uL. Then we ran another agarose gel with this concentrated sample and with another 40 uL from finO gathered sample.

  • This time, finP appeared as a barely visible band, but we were able to purify it, as we did for finO to.

  • We performed purification using Invitrogen's Purelink Quick Gel Extractin Kit, following manufacturer's protocol (Protocol 7) without modifications).

Marcelo

finOP purification results

  • After purification procedure, we quantified total DNA present in both samples.

  • finO resulted in 15 ng/uL. Although very low, this amount might be enough for proceeding in biobrick construction.

  • As for finP, no amount could be measured. =/ Probably, the barely visible band hadn't enough DNA for detection or there was a significant loss during purification process.

Marcelo

finO

  • Even with no longer finP available, we continued finO's work by ligating finO purified sample into biofusion digested vector, according to Protocol 11.

  • We then dialyzed the ligation product for 20 minutes and transformed them into electrocompetent E. coli bacteria, strain DH10B, according to Protocol 3.

  • After one hour being incubated at 37 ºC, transformed cells were plated into LB-AMP media.

  • Plates were incubated at 37 ºC for an O/N period.

Marcelo

YeastGuard

pADH1+YFP

  • The colonies grew up very well in the plates. So, we selected 10 colonies to grow in liquid LB+AMP media O/N.


Wesley and Gleidson


YEP vector

  • Purification of the bands that correspond to the YEP size digested with PvuII. Then we recirculamos the YEP trough a ligation reaction.

Yep.JPG



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