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Team:Valencia/Notebook/July 3week
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July 3rd Week
July 19th
We performed a miniprep in order to purify the plasmid XXXXXXXXX, that was kindly sent by Kausik Li, containing the Aplysia prion XXXXXXXX.
The digestion with BamH1 clearly showed that the purification had been successful. (PHOTO needed).
We also started the protocol for next day's transformation of yeast strain 2606 with the plasmid pLZ, which we already tried last week but obtained no results.
This was also our first day of lab work with PM2 (LEA gene) transformed E.coli strains: In order to separate the PM2 gene from its vector ( pET28 plasmid) we carried out a restriction enzyme digestion of the pET28 plasmid with the PM2 gene following the protocol described below:
1-Addition of 54 µl of DNA.
2-Addition of 6 µl of the idoneous buffer for the restriction enzymes we are going to use.
In this case, it was chose the ¨H¨ buffer.
3- Addition of 1µl of EcoR1 restriction enzyme.
4- Addition of 1µl of Xho1 restriction enzyme.
5- keep the digestion mix at 37º C overnight. These coditions promote the digestion reaction.
In a parallele way one PM2 transformed E.coli strain colony was cultured in a tube filled with 5 ml of LB medium and 5 µl of kanamycin and kept at 37ºC in an stove for the whole night. We will profit this culture for our future pET28 plasmid isolations.
July 20th
The transformation conducted the day before showed different results. The 5523 strain of Saccharomyces cerevisiae grew fine. However, the 2606 strain showed no growth at all. This may be due to the excesive OD of the culture at the beginning of the transformation process (so the yeasts were not at exponential growth phase).
Therefore we repeated the transformation protocol of the 2606 (with LiAc) strain and let the yeast grow at 30ºC during three days.
Word of the day: flowthrough! Sounds awesome, isn´t it!?
BREAKING STORIES: Due to maintenance issues, the air condicioned and the regular devices were off from 3 p.m. on. So we had to find the lab machines plugged in to the emergency system and we found out the true meaning of the word HOT. More soon, on this underground section known as BREAKING STORIES.
