Team:Florida Atlantic/Experiments
Florida_Atlantic
Experiments
Wet Lab Protocols
LB Broth 25g LB Broth, Miller 1L Water Mix LB powder into water, autoclave for 15 minutes. Supplement with antibiotics (Chloramphenicol, 35μL/mL) as needed. LB Agar 25g LB Broth, Miller 15g Granulated Agar 1L Water Mix LB powder and Agar into water. Heat until agar is dissolved and solution is clear, avoid boiling over. Autoclave for 15 minutes. Supplement with antibiotics (Chloramphenicol, 35μL/mL) as needed. Pour 20mL into sterile culture plates and let cool.
Transformation of Competent Cells
50μL Competent Cells (DH5α E. coli) 10ng Plasmid DNA 450μL LB LB/Chloramphenicol Plate Thaw Competent cells on ice and add plasmid DNA. Let sit for 20 minutes on ice. Heat shock cells at 45 o C for 30 seconds and then return to ice for 2 minutes. Add LB and incubate at 37 o C for 2-3 hours. Plate 100μL of the cells on a LB/Chloramphenicol plate and incubate at 37 o C overnight.
Dry Lab Protocols
Protein Reverse Translation Isolate the protein sequence of interest and reverse translate using the E. coli preferred codon library in SnapGene. After reverse translation, look for out-of- frame coding regions and alter the codons so that no transcription is likely to occur. Finally, run a BLASTX protocol to ensure that the nucleotide sequence still encodes the protein of interest.
