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 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 which we 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.

July 23rd

A plasmid isolation protocol was performed in order to purify pET28A (containing PM2 gene) and YAP ( containing PUC-ACT, a yeast promoter) plasmids from the E. coli cultures that we prepared the day before ( see July 22nd ). The steps that were followed were based on the protocol included at the extraction kit that was used.

After these plasmids were isolated, they were digested by the restriction enzymes which allow the proper gene / promoter isolation.

  1. For the digestion of the pET28A plasmid (containing PM2 gene) we used EcoRI and HindIII enzymes. The digestion reaction was buffered by B buffer .
  2. For the digestions of the YAP plasmid (containinf puc-ACT promoter) we used BamHI and SmaI restriction enzymes. The digestion reaction was buffered by A buffer.

In order to analyze the result of both digestions, an agarose gel electrophoresis was run. No bands corresponding to the expected DNA sizes were detected. Unfortunately we will have to repeat the plasmids isolation and subsequent digestion processes.

July 26th

We repeated the plasmid purification that we performed the day before, but modifying the original protocol. In this case, we added an elution buffer which was previously heated at a temperature of 55ºC, as a phd student recommended us. Theoretically it would allow us to yield a 25% more of plasmid DNA than the day before.

Spectrophotometer analysis estimated a concentration of pET28A plasmid of 41.24 ng/µl . YEP plasmid concentration was 29.36 ng/µl. the The ratios of the absorbance at 260 and 280nm (A260/280) were 1.82 for pET28A and 1.79 for YEP. These data showed us that the purity of both plasmid isolations was high, although their concentrations were low. After this plasmid isolation, pET28A-PM2 plasmid was digested with EcoR1 and HindIII restriction enzymes, using the same buffer as the day before.

Before leaving the lab until the next day, we cultured more pUC-ACT and PET-PM2 E. coli colonies in BL liquid medium (procedure and antibiotics used in the medium are show on the July 22nd callendar`s post).

July 27th

The second try of Saccharomyces cerevisiae 2606 strain transformation did not succeed either. Today we conducted a third try taking some precautions in order to find out where is the problem. We prepared three different dishes. In the first one we cultivated the 2606 in a SD medium with aditional ade, trp, his, ura and leu. In the second one we cultivated the 2606 strain and we made in going through the whole transformation protocol but without adding the plasmid pL2/GZ. Finally we prepared a third dish in which we procceed with the transformation normally.

This way we intend to determine in which step the problem is located.

About E. coli and LEA protein: We analyzed the pET28A plasmid digestion of the day before via an agarose electrophoresis. As it can be seen at the picture, the plasmid was in a very low concentration. Due to this negative result we had to repeat the pET28 plasmid isolation procedure. We profited to try again to isolate also the pUC-ACT plasmid. The protocol which was followed was modified, hoping to obtain both plasmids in the proper concentration for their future uses. These points in such protocol were:

  1. It was used all the cell culture that there was left ( 3 ml for both plasmids).
  2. the elution step was performed adding two consecutive aliquots of 25μl of elution buffer at 60ºC.

Spectrophotometer analysis estimated a concentration of pET28A plasmid of 103.01 ng/µl . YEP plasmid concentration was 71,83 ng/µl. the The ratios of the absorbance at 260 and 280nm (A260/280) were 1.96 for pET28A and 2.3 for YEP. These data showed us that the concentration of both plasmids was high, although their purity was a little lower than expected: pET 28A plasmid might be contaminated by proteins. pUC-ACT might be contaminated by proteins and phenol.

After pET-PM2 and pUC-ACT plasmid isolations, they were digested following the same restriction enzymes and buffers (and in the same quantities) that were described in past digestions. The digestions were checked by mean of a preparative electrophoresis (using all the quantity of digested samples). The picture below showed that pET28A-PM2 plasmid digestion appeared as 2 bands of the expected sizes( approx. 5.4 bp for the vector and 1.8 bp for PM2 gene). pUC-ACT digestion appeared as 3 bands instead of the 2 bands that were expected to appear.