October: Second Week

Digestions with different enzymes using several standard procedures were made, followed by ligations. This was the first for the assembling of all the different parts.

Different experiments were carried out to test the new sequences and parts, and inserted into the cells for transformation. Transformed or non-transformed cells were tested under different concentrations of iron, under different environmental conditions.

This week we had several difficult situations to work through and find solutions for. For instance:

1.We had to adjust the concentrations of antibiotic several times until achieving the right one. Thus, we used different concentrations of penicillin for the plasmids until we got it right. We found that the necessary concentration for the penicillin was between 50ug/mL and 100ug/mL. Nonetheless, we used 100ug/mL to avoid false positives. However, we did not have at the moment Xgal stock, which is necessary to make a trial highly effective at lower concentrations of penicillin. As a solution, we prepared the LB solid in Petri dishes with 100ug/mL of penicillin and we used them to grow the cells, which had been transformed the night before (kept at 4 degrees Celsius).

<p style="font-family: Rockwell"2. The time intervals for transformation haven’t been the most favorable, usually, the elapsed time is counted starting from the first transformed sequence (which in our case was positive), and then it is estimated. Since we ran out of competent cells, we had to make more competent cells for stock.</p>

3. Although we tried to use commercial competent cells, imported form USA, we could not use them, because they arrived a week later after shipping, and they have lost their viability. Thus, we had to rely on the cells that we made. This demanded a great deal of time working in the lab.

4. The transformation, cutting, and ligation process is being carried out parallel. The first ligations were performed using XbaI and SpeI, afterwards, the 5’ end of the plasmic DNA were dephosphorilated (in order to prevent the DNA from recircling). In this process we found out that we had to precipitate and separate the DNA by means of extraction using chloroform and precipitation with alcohol. Since we didn’t have saturated phenol, we stopped the reaction with a CIAP stop buffer and we immediately subjected the DNA to a thermal shock in order to avoid re-circularization. This method worked successfully. Afterwards, we purified the sample of enzymes and the buffers from the CIAP phosphatase using a plasmid purification kit from PROMEGA (cat. #A9281). The results are included in the Gel Electrophoresis Report.

5. We had a previous problem where we were running out of cells to transform the plasmids and use the parts. To solve this situation, we divided each tube with cells into 4 tubes and to our surprise, the process has worked, with fewer yields but it has worked.