* A electrophoresis was made with the product of the digestion performed yesterday. Then, we purify the bands corresponding to the size of the pADH1 in the biofusion vector and the YFP part.
+
*<p style=”text-align:justify;”>An electrophoresis was made with the product of the digestion performed yesterday. Then, we purified the bands corresponding to the size of the pADH1 in the biofusion vector and the YFP part.</p>
-
* So we made the ligation of the YFP part in the biofusion vector that contains the ADH1 promoter. Then we transformed this construction in ''E. coli'' that growth over-night in the kanamicin media plate.
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*<p style=”text-align:justify;”>So we made the ligation of the YFP part in the biofusion vector that contains the ADH1 promoter. Then we transformed this construction in ''E. coli'' that has grown O/N in the kanamicin media plate.</p>
* ??Digestão Lisozima (X e S)??
* ??Digestão Lisozima (X e S)??
''Wesley and Gleidson''
''Wesley and Gleidson''
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+
====New strategy: pGEM====
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*<p style=”text-align:justify;”>The plate of ''E. coli'' transformed with the pDLD+Biofusion had only two colonies. We did the colony PCR to find correct constructions of the pDLD and Lysozyme in Biofusion. We found two fragments corresponding to pDLD, but the negative control amplified the same fragment too! We decided to do miniprep of both colonies and re-confirm it by digestion. We found one expected band of lysozyme. We did miniprep of this colony and chose an inespecific one as well.</p>
+
GEL
+
+
*<p style=”text-align:justify;”>JENorf+pGEM grew!! Later that day we found out that we forgot to plate X-Gal =/ We did more plates, this time with X-Gal!! Waste of time!! =(</p>
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*<p style=”text-align:justify;”>We digested the Lysozyme+Biofusion vector and pJEN1+Biofusion vector with ''Xba''I and ''Pst''I to recover the part fragment with one ''Not''I site. The Lysozyme digestion worked so we purified the correct fragment, ligated it in biofusion again in order to recover the second ''Not''I site. Unfortunately the pJEN1 digestion didn’t work.</p>
+
GEL
+
+
*<p style=”text-align:justify;”>We inoculated pJEN1+biofusion and pDLD+biofusion in liquid media to do miniprep tomorrow.</p>
Transformation of the BBa K112806 + BBa B0015 ligation
We purified the digestion from agarose gel and use the product to do a ligation with BBa B0014 + BBa K112806 according to Protocol 11.
The ligation was used in the transformation of E. coli. We hope with BBa B0014 the digestion is better than B0015.
finO and finP with pGEM - confirmation
Once we confirmed that several samples actually have finO and finP inserts ligated into pGEM vector (by the digestion procedure performed yesterday), we could proceed to the final step on confirming our finOP-pGEM ligations.
We performed a PCR for each miniprep samples we got, using a specific forward primer for our inserts (finO and finP) and a reverse primer that is specific for pGEM vector (primer M13, see pGEM manual). This will allow us to verify that we have the insert ligated into the plasmid and, further more, will allow us to check whether or not our inserts are indeed in the correct frame position.
According to the pictures, both finO and finP's PCR resulted in bands that reached the expected size in several samples.
Therefore, we concluded we sucessfully have finO and finP correctly ligated into pGEM vector. Next step is ligating them into biobrick's vector.
Marcelo
YeastGuard
pADH1+YFP
An electrophoresis was made with the product of the digestion performed yesterday. Then, we purified the bands corresponding to the size of the pADH1 in the biofusion vector and the YFP part.
So we made the ligation of the YFP part in the biofusion vector that contains the ADH1 promoter. Then we transformed this construction in E. coli that has grown O/N in the kanamicin media plate.
??Digestão Lisozima (X e S)??
Wesley and Gleidson
New strategy: pGEM
The plate of E. coli transformed with the pDLD+Biofusion had only two colonies. We did the colony PCR to find correct constructions of the pDLD and Lysozyme in Biofusion. We found two fragments corresponding to pDLD, but the negative control amplified the same fragment too! We decided to do miniprep of both colonies and re-confirm it by digestion. We found one expected band of lysozyme. We did miniprep of this colony and chose an inespecific one as well.
GEL
JENorf+pGEM grew!! Later that day we found out that we forgot to plate X-Gal =/ We did more plates, this time with X-Gal!! Waste of time!! =(
We digested the Lysozyme+Biofusion vector and pJEN1+Biofusion vector with XbaI and PstI to recover the part fragment with one NotI site. The Lysozyme digestion worked so we purified the correct fragment, ligated it in biofusion again in order to recover the second NotI site. Unfortunately the pJEN1 digestion didn’t work.
GEL
We inoculated pJEN1+biofusion and pDLD+biofusion in liquid media to do miniprep tomorrow.
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