SDS-PAGE and Western Blotting DNA Fragment Recovery Bacterial ...
Undergraduate Category: Physical and Life Sciences Degree Level: Bachelor of Science Abstract 268
Freshmen Delving into the Mysteries of Biochemistry Research S. Braun, T. J. Cawley, M. R. Del Mastro, S. G. DiLucia, N. Ganci, G. M. Grifferty, E. M. Hegermiller, S. P. G. Moore, and P. R. Strauss
24 hpf sample
8 hpf sample
Protein 3
Determination of the equivalence point of a titration of 0.5 M disodium phosphate with 0.5 M HCl through first derivative analysis enabled calculation of pKa.
Digestion of DNA Figure 2: Gel Electrophoresis Visualized under UV Light
MW standard
Dra III + Xho I Double Digest
Xho I single digest
Mock digest
MW standard
A) DNA digest with DraIII and XhoI resolved through gel electrophoresis B) DNA fragment excised from agarose gel C) Excised DNA fragment and previously digested DNA fragments resolved via gel electrophoresis D) Excised fragment and corresponding fragment from original digest selected for ImageJ analysis E) ImageJ generated peaks corresponding to relative density of DNA bands, enabling quantification of recovery
Bacterial Transformation and Miniprep of DNA
Figure 4: Plating of Transformed Bacteria and Verification of Miniprep Effectiveness through Gel Electrophoresis
A)
Protein 4
The Coomassie blue stain bound nonspecifically to proteins on the polyacrylamide gel. This allowed visualization of the full quantity of protein present in the embryonic sample.
Figure 6: Western Blot of Beta-Tubulin Visualized by Chemiluminescence
D)
B)
5. Restriction digest of DNA and fragment recovery (Figure 3)
Western Blot analysis of zebrafish embryos revealed the presence of beta-tubulin in both 8 hpf (hours post fertilization) and 24 hpf embryonic samples.
6. Transformation of bacteria and DNA minipreps (Figure 4)
C)
7. SDS-PAGE and western Blotting (Figures 5 and 6) 8. PCR and subcloning of Zebrafish histone H2B variant genes – In Progress!
E)
Figure 5: Bulk Protein Detection by Coomassie Blue Staining of Polyacrylamide SDS Gel
Protein 2
Dra III single digest
4. Restriction digest of DNA (Figure 2)
D)
C)
Protein 1
2. Preparation of solutions 3. Titrations of solutions (Figure 1)
B)
24 hpf sample
1. Pipette calibration
A)
8 hpf sample
Techniques Learned
Figure 1: Titration of 0.5 M Disodium Phosphate with 0.5 M HCl
Figure 3: Resolving Fragments by Gel Electrophoresis, Fragment Extraction, and Quantification of Results
MW Standard
Freshman biology and biochemistry students possess an enthusiasm and curiosity for research that is often overshadowed by their lack of hands-on laboratory experience. Through her directed study program, Professor Strauss offers eager freshmen the opportunity to apply their theoretical knowledge and develop invaluable laboratory skills. By conducting weekly experiments and working in close conjunction with laboratory personnel, students are also exposed to the principles of lab etiquette and professional scientific writing. Students leave the laboratory equipped with the foundation of knowledge and skills they need to pursue their own novel research and contribute to the scientific community. At the end of the semester, students will be investigating how variants of histone H2B impact zebrafish embryological development and will compare the results to embryological development after knockdown of a critical protein involved in repair of oxidative damage to DNA.
Titrations of Solutions
SDS-PAGE and Western Blotting
MW standard
Abstract
DNA Fragment Recovery
The CS2+ plasmid was digested with the DraIII and XhoI restriction enzymes, the fragments were resolved via gel electrophoresis, and molecular weights were determined for each fragment.
A) Untransformed Esherichia coli (E. coli) displayed growth on nonampicillin plate. B) E. coli transformed with pCS2+ unexpectedly displayed no growth on ampicillin plate. Linearization of the plasmid stock was suspected. C) E. coli transformed with pUC19 showed growth on ampicillin plate. D) The efficacy of a DNA miniprep purifying pCS2+ harvested from a successful E. coli transformation was confirmed through gel electrophoresis.
Conclusion This experience gives freshman biology and biochemistry majors invaluable hands-on laboratory experience. The small group setting allows each student to receive the individual attention they need to improve their scientific writing and overall laboratory competence. As students are evaluated holistically, they are given the opportunity to learn in a supportive environment fostered by constructive criticism. The knowledge gained through the directed study will allow the students involved to actively engage in scientific research.
Acknowledgments
The authors would like to thank Nicole Kaplan, Todd Estabrook, Josh Kruchten, and Maureen McGill. Special thanks is extended to Stephen Moore and Professor Phyllis Strauss for their patience and guidance and for making this experience possible.
Freshmen Delving into the Mysteries of Biochemistry Research S. Braun, T. J. Cawley, M. R. Del Mastro, S. G. DiLucia, N. Ganci, G. M. Grifferty, E. M. Hegermiller, S. P. G. Moore, and P. R. Strauss
24 hpf sample
8 hpf sample
Protein 3
Determination of the equivalence point of a titration of 0.5 M disodium phosphate with 0.5 M HCl through first derivative analysis enabled calculation of pKa.
Digestion of DNA Figure 2: Gel Electrophoresis Visualized under UV Light
MW standard
Dra III + Xho I Double Digest
Xho I single digest
Mock digest
MW standard
A) DNA digest with DraIII and XhoI resolved through gel electrophoresis B) DNA fragment excised from agarose gel C) Excised DNA fragment and previously digested DNA fragments resolved via gel electrophoresis D) Excised fragment and corresponding fragment from original digest selected for ImageJ analysis E) ImageJ generated peaks corresponding to relative density of DNA bands, enabling quantification of recovery
Bacterial Transformation and Miniprep of DNA
Figure 4: Plating of Transformed Bacteria and Verification of Miniprep Effectiveness through Gel Electrophoresis
A)
Protein 4
The Coomassie blue stain bound nonspecifically to proteins on the polyacrylamide gel. This allowed visualization of the full quantity of protein present in the embryonic sample.
Figure 6: Western Blot of Beta-Tubulin Visualized by Chemiluminescence
D)
B)
5. Restriction digest of DNA and fragment recovery (Figure 3)
Western Blot analysis of zebrafish embryos revealed the presence of beta-tubulin in both 8 hpf (hours post fertilization) and 24 hpf embryonic samples.
6. Transformation of bacteria and DNA minipreps (Figure 4)
C)
7. SDS-PAGE and western Blotting (Figures 5 and 6) 8. PCR and subcloning of Zebrafish histone H2B variant genes – In Progress!
E)
Figure 5: Bulk Protein Detection by Coomassie Blue Staining of Polyacrylamide SDS Gel
Protein 2
Dra III single digest
4. Restriction digest of DNA (Figure 2)
D)
C)
Protein 1
2. Preparation of solutions 3. Titrations of solutions (Figure 1)
B)
24 hpf sample
1. Pipette calibration
A)
8 hpf sample
Techniques Learned
Figure 1: Titration of 0.5 M Disodium Phosphate with 0.5 M HCl
Figure 3: Resolving Fragments by Gel Electrophoresis, Fragment Extraction, and Quantification of Results
MW Standard
Freshman biology and biochemistry students possess an enthusiasm and curiosity for research that is often overshadowed by their lack of hands-on laboratory experience. Through her directed study program, Professor Strauss offers eager freshmen the opportunity to apply their theoretical knowledge and develop invaluable laboratory skills. By conducting weekly experiments and working in close conjunction with laboratory personnel, students are also exposed to the principles of lab etiquette and professional scientific writing. Students leave the laboratory equipped with the foundation of knowledge and skills they need to pursue their own novel research and contribute to the scientific community. At the end of the semester, students will be investigating how variants of histone H2B impact zebrafish embryological development and will compare the results to embryological development after knockdown of a critical protein involved in repair of oxidative damage to DNA.
Titrations of Solutions
SDS-PAGE and Western Blotting
MW standard
Abstract
DNA Fragment Recovery
The CS2+ plasmid was digested with the DraIII and XhoI restriction enzymes, the fragments were resolved via gel electrophoresis, and molecular weights were determined for each fragment.
A) Untransformed Esherichia coli (E. coli) displayed growth on nonampicillin plate. B) E. coli transformed with pCS2+ unexpectedly displayed no growth on ampicillin plate. Linearization of the plasmid stock was suspected. C) E. coli transformed with pUC19 showed growth on ampicillin plate. D) The efficacy of a DNA miniprep purifying pCS2+ harvested from a successful E. coli transformation was confirmed through gel electrophoresis.
Conclusion This experience gives freshman biology and biochemistry majors invaluable hands-on laboratory experience. The small group setting allows each student to receive the individual attention they need to improve their scientific writing and overall laboratory competence. As students are evaluated holistically, they are given the opportunity to learn in a supportive environment fostered by constructive criticism. The knowledge gained through the directed study will allow the students involved to actively engage in scientific research.
Acknowledgments
The authors would like to thank Nicole Kaplan, Todd Estabrook, Josh Kruchten, and Maureen McGill. Special thanks is extended to Stephen Moore and Professor Phyllis Strauss for their patience and guidance and for making this experience possible.
