Team:UNICAMP-Brazil/Notebooks/September 7

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ColiGuard

finO, finP and pSB1A3 digestion

Now that we have purified samples of the finO and finP sequences (see September 6th for more details), and once we could also recover the pSB1A3 vector (August 10th), it's time to start the construction of our novel biobricks, which will be responsible for inhibiting the conjugation process in our labor cells model (see project overview for further information).
In order to achieve this objective, we permorfed today the digestion with XbaI and SpeI restriction enzymes of the purified sequences finO and finP, and also of the vector pSB1A3. The digestion lasted 3 hours.
After that, we run an agarose gel of the digested product aiming the confirmation of the assay. According to the gel, the vector pSB1A3 was succesful digested. As for the sequences finO and finP, it's not possible to assure whether they were digested or not since the gel resolution does not differentiate just a few base pairs (excised by digestion). Therefore, we need to ligate those sequences into the digested vector in order to confirm that they both were also digested.
FinO appeared in the gel, altough not very visile. As for finP, no visible band was found. We tought that this was kind of expected since finP's band was very weak even before the digestion procedure, and we didn't ran enough volume.

Marcelo

PCR: Cre-Recombinase without ATG

<p style=”text-align:justify;”>After the realization of the miniprep to isolate the Cre-Recombinase, we realized a PCR reaction to amplify the Cre-Recombinase directly from the plasmid, without the amplification of its start codon (ATG). We need the Cre-Recombinase without the ATG codon to the development of one of our devices, being it a new biobrick to be created by our group.
Today we couldn't realize the reaction because we got the wrong primers. ¬¬

Víctor

YeastGuard

WIKI

Today we worked on updating the wiki!

Lactate Permease (JEN1 ORF)

Considering that we didn´t have a specific band in the digestion from the JEN1, we decided to repeat the PCR (we think it was not totally specific) and to purify the correct band. So today we repeated the PCR for the JEN1 ORF.

@@ Line 10: / Line 10: @@
 *<p style=”text-align:justify;”>Now that we have purified samples of the finO and finP sequences (see September 6th for more details), and once we could also recover the pSB1A3 vector (August 10th), it's time to start the construction of our novel biobricks, which will be responsible for inhibiting the conjugation process in our labor cells model (see project overview for further information).</p>
 *<p style=”text-align:justify;”>In order to achieve this objective, we permorfed today the digestion with XbaI and SpeI restriction enzymes of the purified sequences finO and finP, and also of the vector pSB1A3. The digestion lasted 3 hours.</p>
-*<p style=”text-align:justify;”>After that, we run an agarose gel of the digested product aiming the confirmation of the assay. According to the gel, the vector pSB1A3 was succesful digested. As for the sequences finO and finP, both of them appeared in the gel, but it's not possible to assure that they indeed were digested since the gel resolution does not differentiate just a few base pairs (excised by digestion). Therefore, we need to ligate those sequences in the digested vector in order to confirm that they both were also digested.</p>
+*<p style=”text-align:justify;”>After that, we run an agarose gel of the digested product aiming the confirmation of the assay. According to the gel, the vector pSB1A3 was succesful digested. As for the sequences finO and finP, it's not possible to assure whether they were digested or not since the gel resolution does not differentiate just a few base pairs (excised by digestion). Therefore, we need to ligate those sequences into the digested vector in order to confirm that they both were also digested.</p>
+*<p style=”text-align:justify;”>FinO appeared in the gel, altough not very visile. As for finP, no visible band was found. We tought that this was kind of expected since finP's band was very weak even before the digestion procedure, and we didn't ran enough volume.
 ''Marcelo''