Time goes by... (El tiempo pasa...)

Follow us:

Our main sponsors:

Our institutions:

Visitor location:

July 4th Week

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.