We discussed and designed our experiment!
a. LB preparation: The LB preparation procedure included 1g tryptone, 1g NaCl, 0,5g Yeast extract in a Duran bottle. Then 100mL of deionized water were added and consequently blended with a magnetic bead. When they got dissolved, we measured the pH and add NaOH until pH=7 (magnet removed). We sterilized it for 20 minutes and stored it in room temperature (25 oC).
b. Bacteria transformation: We cultured dH5a E. coli in a TS broth for a whole day in a cold 1.5 mL tube and on ice and added 1 uL pcDNA3.1 plasmid into the dh5a-cultutred tube. After minimal shaking, we left the dh5a-cultured tube alongst wiith the pcDNA3.1 plasmid on ice for 20 minutes. We performed a heat shock at 42 oC for 30 seconds on the pre-prepared heatblock machine with the respective temperature. We left the dh5a-cultured tube for 5 minutes on ice and added 200 uL of room temperature LB. We incubated it at 37 oC for 1 hour, and allowed an overnight growth.
a. Plasmid purification: We utilized the Nucleospin Plasmid (Nucleospin, Macherey-Nagel) in order to isolate the pcDNA-3.1 plasmid as well as assessed its purity and concentration via Q500 UV-Vis Spectrophotometer (Quawell). Derived concentration was measured at 30 ng/uL, which was expected due to the limited culture quantity (we used 200 uL instead of the recommended 1- 5mL \riotgamesculture quantity).
b. Plasmid DNA electrophoresis: We diluted 0,3 g Agarose and added it to 30 ml TAE buffer. After partial dilution, we added 1 uL Ethidium Bromide and poured it into the electrophoresis base and cooled it down. We used 2 ul of 100 bp Ladder and placed it in the first channel. We added 5 ul of plasmid DNA, 1 ul of water and 1 ul of Loading Dye, consisting of Glycerol and appropriate for the naked eye dyes. After that, we placed into the second channel and did the electrophoresis at 100V for 35 minutes. We understood that the DNA was fragmented so we decided to redo the whole process.
a. Experimental redesign: we decided to create solid cultures and add Ampicillin as a restriction antibiotic for transformed cell selection.
b. LB preparation: we prepared a new LB solution to be sure of its quality. We also prepared 100 mL LB Agar using 1,5 g Agar.
c. Pedri dishes preparation: we poured the LB Agar into 4 pedri dishes.
a. Restriction antibiotic addition: we created an Ampicillin solution with a concetration of 0,55 mg/ml and added it to the LB Agar (after we had melted it using heat) until a final concentration of 50 μg/ml.
b. Bacteria transformation: We placed the iced and stored at -80 oC with glycerol cultured dH5a E. coli in TS broth in a cold 1,5 mL tube on ice and added 1 uL pcDNA3.1 (3 ng/ul) plasmid into the 1,5 mL tube. After soft shakening, we left the dh5a-cultured tube alongst with the pcDNA3.1 plasmid on ice for 30 min. We performed a heat shock at 42 oC for 45 seconds on the machine that we had already turned and left it for 5 minutes on ice. We added 800 uL of room temperature LB. We incubated it at 37 oC for 1 hour, and left it for overnight growth.
c. LB Agar plates layaring: we layared the Ampicillin-rich LB Agar onto the plates and left overnight to be solidified again.
a. Experimental redesign: after we observed that the bacteria culture in the LB Broth was not hazy at all, we realized that the recultured E. coli had lost their competency and none of them grew due to Ampicillin presence. So we decided to use the stock, compentent prepared E. coli cells.
a. Bacteria transformation: we used the original tube with non-cultured E. coli cells from NEB, put it on ice and added 1 uL of pcDNA3.1 (3 ng/ul) plasmid into it. After soft shakening, we left it on ice for 30 min. We performed a heat shock at 42 oC for 45 seconds on the machine that we had already turned on and prepared the temperature and left it for 5 minutes on ice. We added 800 uL of room temperature LB. We incubated it at 37 oC and at shaking for 1 hour. 600 ul of the 1 ml of transformated bacteria were mixed with 2,05ml LB and 0,2ml Ampicillin. Of the remaining 400 uL of dh5a-cultured E. coli, half of those (200 ul) were used as a control culture with 0,75 ml LB, while the rest (200μl) were added into a solid culture; the latter were also split into two different concentrations (150 ul and 50 ul).
b. Solid culture layaring: We layared the transformated bacteria onto the LB Agar using a glass triangle and stored them at 37 oC overnight.
a. Plasmid DNA purification: We utilized the Nucleospin Plasmid (Nucleospin, Macherey-Nagel) for 200 ul of the E. coli culture and determined the concentration at 14 ng/ul via the Q500 UV-Vis Spectrophotometer (Quawell) which was considerably low.
b. Electrophoresis: we followed the same protocol, nevertheless no results were derived, probably due to the low concentration.
c. Control culture modification: we poured the control blurry liquid culture onto 2 LB Agar dishes and left it for overnight growth.
a. Bacteria reculture: we took colonies from the solid culture and placed them in three 5 ml tubes with LB and left them overnight at 37 oC to grow.
a. Plasmid DNA purification: using the NucleoSpin Plasmid kit (Macherey-Nagel) we isolated the plasmid and calculated its concentration in nanodrop at 16 ng/ul via Q500 UV-Vis Spectrophotometer (Quawell).
b. Electrophoresis: we used the electrophoresis protocol and observed a light band, respective to the desired plasmid length.
a. Plasmid DNA and gene digestion: At first, 10 ul of the plasmid and 2 ul of the compatible for both enzymes IV buffer, 6 ul of deionized H2O and after having them out of the ice, we added 1 ul of each restriction enzyme (EcoRI and XbaI) to a final volume of 20 ul. We left it for incubation at 37 oC for 1 hour.
b. Electrophoresis: we used the electrophoresis protocol and observed that the plasmid was not digested in contrast to the gene under study.
c. Experimental redesign: we decided to change the protocol and use an increased volume of the plasmid that had a small concentration.
d. Second plasmid DNA digestion: we used 20 ul of the plasmid, 1,5 ul of each enzyme (EcoRI and XbaI), 3 ul of the IV buffer and 4 ul of H20 (30 ul in total). We left it again at 37 oC for 1 hour.
e. Second electrophoresis: we confirmed that the digestion was succesfull.
a. Ligation: We diluted 1,6 ul of the DNA insertion sequence, 3,3 ul of the plasmid vector, 2 ul of the high-T4 ligase buffer, 1 ul of the T4 Ligase into 11.7 ul of purified H2O. We mixed everything gently by pipetting up and down and we microfuged it for a bit. Because we used cohesive ends, we incubated at 45 oC for 10 minutes according to the high T4-ligase protocol. We heated it at 65 oC for 10 minutes in order to inactivate the ligase according to the manufacturer’s protocol, and consequently put it on ice to prepare it for the next step.
b. PCR: as a way to confirm that the ligation procedure had been succesfully conducted, we performed a Polymerase-Chain-Reaction (PCR). In specific, we designed two appropriate oligonucleotide primers that binded on either site of the recognition sites. Hence, a recombinant plasmid would yield a larger PCR product due to the presence of the inserted DNA part. We stored the PCR products at 4oC.
a. Electrophoresis : we performed the appropriate protocol and managed to depict the recombinant DNA that is provem by its different position than the non-recombinant on the gel.
a. Bacteria transformation: we followed the tested protocols and left the bacteria at 37oC overnight to grow.
a. Results: we saw that we had succesfully grown bacteria with the recombinant plasmid.