Source Of The Recombinant DNA Polymerase SK72 Lab Report
3.1 Source of the Recombinant DNA Polymerase SK72
The recombinant E. coli BL21 (DE3) containing the pET 32b/ DNA polymerase SK72 gene was provided by the Laboratory of Enzyme and Microbial Technology, Institute of Bioscience, Universiti Putra Malaysia.
3.2 Preparation of the Lysogeny Broth (LB) Agar Plates
LB agar plates were prepared by dissolving 10g tryptone, 5g yeast extract, 5g NaCl and 15g bacterial agar in 950mL of deionized water. The pH of the medium was adjusted to 7.5 using 1M NaOH before bringing the volume up to 1L. After autoclaving at 121oC for 15 minutes, the medium was cooled to approximately 55oC before 100ug/mL ampicillin was added. The medium was then poured into the petri dishes to let them harden before inverting and storing …show more content…
Prior to use, the column was equilibrated using 20mM sodium phosphate buffer (pH6.5). The column was washed for 10 CV with the same buffer after the protein loading and the bound proteins were eluted with a linear gradient of NaCl (0-0.5M) in the same buffer over 10 CV at a flow rate of 1mL/min. The negative ions (Cl-) in the salt solution were competed with protein in binding to the resin. Fractions of 2mL were collected and the purity was assessed by 10% SDS-PAGE.
3.5 Determination of the Concentration of the DNA Polymerase SK72
The amount of protein was measured using the commercialized Bradford reagent as described by Bradford (1976). A standard curve was plotted in a range 100-1000ug/mL in a Bradford-compatible buffer using bovine serum albumin as substrate (Appendix A). 1mL of the dye solution was added to 20uL of the protein sample, mixed and incubated for 10 minutes at room temperature before measuring the absorbance at 595nm using the spectrophotometer.
3.6 Verification for the Homogeneity and Molecular Weight of the DNA Polymerase …show more content…
YASARA program was used to arrange the backbone of the DNA polymerase SK72 identically to the known 3D selected template, which was chosen by virtue of having the highest sequence identity with the targeted sequence. After homology modelling, the model refinement and evaluation of the model quality for correctness of the overall fold and stereochemical parameters like bond lengths and angles was done using Verify3D, Errat Plot and Ramachandran Plot (Bowie et al., 1991 and Colovos et al., 1993). The putative model was used to analyze and interpret the structure, the active site and the special feature of the protein (Ma et al.,
The recombinant E. coli BL21 (DE3) containing the pET 32b/ DNA polymerase SK72 gene was provided by the Laboratory of Enzyme and Microbial Technology, Institute of Bioscience, Universiti Putra Malaysia.
3.2 Preparation of the Lysogeny Broth (LB) Agar Plates
LB agar plates were prepared by dissolving 10g tryptone, 5g yeast extract, 5g NaCl and 15g bacterial agar in 950mL of deionized water. The pH of the medium was adjusted to 7.5 using 1M NaOH before bringing the volume up to 1L. After autoclaving at 121oC for 15 minutes, the medium was cooled to approximately 55oC before 100ug/mL ampicillin was added. The medium was then poured into the petri dishes to let them harden before inverting and storing …show more content…
Prior to use, the column was equilibrated using 20mM sodium phosphate buffer (pH6.5). The column was washed for 10 CV with the same buffer after the protein loading and the bound proteins were eluted with a linear gradient of NaCl (0-0.5M) in the same buffer over 10 CV at a flow rate of 1mL/min. The negative ions (Cl-) in the salt solution were competed with protein in binding to the resin. Fractions of 2mL were collected and the purity was assessed by 10% SDS-PAGE.
3.5 Determination of the Concentration of the DNA Polymerase SK72
The amount of protein was measured using the commercialized Bradford reagent as described by Bradford (1976). A standard curve was plotted in a range 100-1000ug/mL in a Bradford-compatible buffer using bovine serum albumin as substrate (Appendix A). 1mL of the dye solution was added to 20uL of the protein sample, mixed and incubated for 10 minutes at room temperature before measuring the absorbance at 595nm using the spectrophotometer.
3.6 Verification for the Homogeneity and Molecular Weight of the DNA Polymerase …show more content…
YASARA program was used to arrange the backbone of the DNA polymerase SK72 identically to the known 3D selected template, which was chosen by virtue of having the highest sequence identity with the targeted sequence. After homology modelling, the model refinement and evaluation of the model quality for correctness of the overall fold and stereochemical parameters like bond lengths and angles was done using Verify3D, Errat Plot and Ramachandran Plot (Bowie et al., 1991 and Colovos et al., 1993). The putative model was used to analyze and interpret the structure, the active site and the special feature of the protein (Ma et al.,