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ColiGuard
Cre-Recombinase without ATG + terminator
Now that we have the Cre-Recombinase biobrick, our new plan is to assemble its device. We first decided to add the terminator region BBa_B0015 to the Cre-Recombinase without ATG.
We performed the digestion from both terminator and the plasmids that have our biobrick (see October 5, 9 and 10). The Cre's digestion was performed with EcoRI and SpeI and the digestion of terminator with EcoRI and XbaI, both incubated at 37°C for 3 hours.
We ran an 1% agarose gel and observed that the digestion of the terminator ocurred as expected, but not the plasmids containing Cre-Recombinase. What happened?
Victor
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.
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
PY Promoter - Ligation reaction and transformation
Fabi and Léo
YeastGuard
New strategy: pGEM
We did miniprep of pJEN1+Biofusion and pDLD+Biofusion (without the final NotI site), digested the plasmids with XbaI and PstI, ligated the digested fragment with biofusion again, in order to recover the second NotI site. Then we transformed competent E. coli and plated in LB+Amp media.
Raíssa and Taís
pADH1+YFP
Wesley and Gleidson
YEP358
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