Team:Valencia/Notebook/July 3week

From 2010.igem.org

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(July 22nd)
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==July 22nd==
==July 22nd==
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We detected individual colonies on the two PM2 gene transformed ''E.coli'' solid cultures that we prepared the day before. This positive result allowed us to transfer one of the colonies into a  tube with LB medium and Kn. We also took one colony of an ''E.coli'' culture transformed with a YAP plasmid containing a yeast promotor which arrived to the lab today, and transferred it to a tube with LB liquid medium and Ampicillin.
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We detected individual colonies on the two PM2 gene transformed ''E.coli'' solid cultures that we prepared the day before. This positive result allowed us to transfer one of the colonies into a  tube with LB medium and Kn. We also took one colony of an ''E.coli'' culture transformed with a YAP plasmid containing a yeast promotor which arrived to the lab today, and transferred it into one tube with LB liquid medium and Ampicillin.
We finally maintained both cultures at 37ºC in a stove for 16 hours.
We finally maintained both cultures at 37ºC in a stove for 16 hours.

Revision as of 17:13, 28 July 2010

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Contents

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 the obtention of a growth curve.

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.

LEA&E.coli: As it can be read above ( JUly 19th) , we constructed a cellular growth curve of our E. coli culture. In order to do so we got a dilution of the 25% of the original culture. This diluted cell culture was kept in a stove at 37ºC along all our experiment. We measured the optical density ( wavelength=600nm) every 30 minutes until we detected that our population of cells reached the steady state rate of growth. The growth curve graph is shown in the ¨Gallery¨.

We also checked the digestion we prepared the day before by mean of a preparative electrophoresis using all the quantity of sample we had ( 55µl). We observed the expected DNA bands but unfortunately they were companied by many other bands. This result suggested that we failed in our digestion.

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.

July 21st

A transformation of E.coli competent cells with pET28A plasmid carrying PM2 gene was performed. Our goal was to isolate the named plasmid in order to repeat the digestion of the day before. The protocol followed was based on a heat shock procedure :

  1. Addition of 2μL of pET28A plasmid to 100μL of a dilution of competent cells and storage of the mix for 27 minutes in ice.
  2. Heat shock: Treat the cells+plasmid mix with a temperature equal to 42ºC for 90 seconds. After that, move the mix to a cube filled with ice and leave it there for 5 minutes.
  3. Culture 103 μL of the mix in LB liquid medium and keep it in a stove at 37ºC for 85 minutes.
  4. Culture two different aliquots ( seriated dilutions: 0.1 and 0.01) of transformed cells on two independent LB+IPTG+Xgal+Kn solid medium plates and keep them in a stove at 37ºC for the whole night.

July 22nd

We detected individual colonies on the two PM2 gene transformed E.coli solid cultures that we prepared the day before. This positive result allowed us to transfer one of the colonies into a tube with LB medium and Kn. We also took one colony of an E.coli culture transformed with a YAP plasmid containing a yeast promotor which arrived to the lab today, and transferred it into one tube with LB liquid medium and Ampicillin. We finally maintained both cultures at 37ºC in a stove for 16 hours.