Previous Weeks Summary:
In the first week of lab, we were told to research Alpha-B-crystallin which is a protein found in the lens. In order to find the mRNA sequence and amino acid sequence of Alpha-B-crystallin, the NCBI protein database was used. Followed by this we utilized the Expasy translation tool to reveal our nucleotide sequence of codons from the mRNA sequence. Our start codon was methionine and our stop codon was lysine. We were assigned to create a mutation of the 159th residue which involved mutating an isoleucine to a leucine. We developed a primer using the Quickchange primer design that involved mutating the first and third base. The primers were ordered from Invitrogen Custom Primers.
For the second week in lab we focused on preparing LB agar (with antibiotics added to ensure that the bacteria, that did not take up the plasmid, will not grow) as well as LB media. We also spent a majority of our time preparing for a PCR by mixing contents including: forward and reverse primers, template DNA, and Q5 Hot Start High-Fidelity 2X Master Mix (which contains a polymerase with 3’->5’ exonuclease activity, dNTPs, and Mg2+). Towards the conclusion of lab, the PCR tubes containing the contents necessary for a successful PCR were added to a thermocycler. The thermocycler’s role is to provide strict control over the reaction temperature and duration of exposure to each distinct temperature, this ensures the amplification process. We also poured the prepared LB agar into petri dishes to be stored in the fridge as well as leaving our LB media in its container at our lab benches until further use for the upcoming weeks in lab. For this past week in lab transformation was carried out. The first thing we did was to add 1 µl of our PCR product, 5 µl of 2X KLD reaction buffer, 1 µl of 10X KLD enzyme mix and 3 µl of nuclease free water to a 0.6 ml micro tube. after the KLD Reaction, which involved the mixing of PCR product 2X KLD Reaction Buffer, 10X KLD Enzyme Mix, and Nuclease-free water. The KLD enzyme mix contains a mixture of kinase, ligase and Dpnl enzymes in a buffer that preserves the activity of the enzymes. This blend provides intramolecular ligation, circularization, efficient phosphorylation and template removal in a single quick reaction at room temperature. Followed by this we allowed the contents in the micro tube to sit at room temperature for 5 minutes while we prepared our ice bath. Then our instructor gave us 3 samples on BL 21 and one sample of 5 to place in the ice bath. We then prepared 3 Eppendorf tubes within the ice bath with 50 µl of the BL 21 each and 1 Eppendorf tube with 50 µl of the 5 cells. One of the BL 21 tubes had no plasmid and that served as a negative control. Another BL 21 tube had a wild type plasmid to serve as a positive control and the last BL 21 tube contained our plasmid. The 5 tube also contained our plasmid. The negative control was used to portray no growth and the positive control was used to show growth. After all 4 Eppendorf tubes sat in the ice bath for 30 minutes we heat shocked at roughly 44 C for 30 seconds and then incubated in ice for another 5 minutes. We then transferred the contents in the 4 Eppendorf tubes to a larger micro tube before adding 950 µl of LB media to each micro tube and then allowing the contents to shake at 37 C for 1 hour. After this we spread a 100 µl of the contents from each micro tube to our petri dishes containing the LB agar we prepared last week and incubated these petri dishes at 37 C over night.