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Internship Progress Review

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Internship Progress Review

  1. 1. Internship Progress Report By Kailash Jayachandran 16INT0001
  2. 2. Background  Student at Pennsylvania State University (Class of 2019).  Majoring in Biochemistry & Chemical Engineering.  Did research in Environmental engineering.  Currently a student mentor.  Primarily interested in research & have a passion for it.  Doing an internship at VIT to further enhance myself.  Working in the labs of Dr. Karthikeyan, Dr. Gothandam, Dr. Rasool & Dr. Babu at SBST.  Learning various techniques under the tutelage of research scholars.
  3. 3. List of Techniques  Inoculation & Streaking of cultures- Quadrant & Zig- Zag streaking  Polymerase Chain Reaction (PCR)  Agarose Gel Electrophoresis  Principles of HPLC  Bacterial Genomic DNA isolation  Bacterial Plasmid DNA isolation  SDS PAGE  Immunofluorescence  Western Blotting  Principles of ELISA
  4. 4. Inoculation & Streaking of Cultures  Generally used to grow specific bacteria in a nutrient broth  This is generally done to isolate colonies by specificity.  Factors of growth- Type of agar, conditions & composition of broth.  Microbiological techniques- Sterilisation, aseptic techniques, inoculation and incubation.  Streaking techniques- Quadrant & Zig- Zag streaking Quadrant StreakPlate Streak
  5. 5. Polymerase Chain Reaction (PCR)  PCR is a technique which is used to amplify DNA & generate millions of copies of that specific DNA sequence.  It has 3 steps- Denaturation of the double-stranded DNA into a single- stranded molecules; annealing of the primers to the specific area of interest and an extension phase.  Required materials- DNA substrate, forward & reverse primer, Taq Polymerase, dNTP’s, Buffer solution and divalent cations ( Mg2+ ).  Three types of PCR used in the lab- Gradient, Real time & Reverse transcriptase PCR.  Used in medical & biological research (genetic diseases & forensics) PCR full process
  6. 6. Agarose Gel Electrophoresis  Separation of the PCR product/ DNA strands is required.  This is done by using an electric field to move DNA towards the anode & they move accordingly based on length (Charge/size).  Required materials- Agarose solution, TAE buffer & Ethidium Bromide.  Thus, dyed samples are added to the casted gel.  Visualization is done via UV transiluminator or Gel doc. Visualization via UV Visualizing gradient PCR product under UVGel Electrophoresis
  7. 7. Principles of HPLC  HPLC is used to separate, identify & quantify components dissolved in a liquid solvent with a high analytical resolution.  HPLC pumps the sample in the mobile phase at high pressure through a column filled with chromatographic material ( stationary phase).  Gas stream of helium & nitrogen help in facilitating sample transport.  So, compounds present in trace amounts can also be measured.  Sample retention time will vary depending on the interaction between the stationary phase, the molecules being analyzed, and the solvent, or solvents used.  Interaction between both columns is at different rates. So, this will impact the exit time of the sample. HPLC Machine HPLC Mechanism
  8. 8. Bacterial Genomic DNA Isolation  Bacterial cells are grown in a medium till they reach log phase & are centrifuged.  Then, resuspension is done by adding lysis solution & Proteinase K is used to degrade the gram negative bacterial cell wall.  Cell lysis is followed & ethanol is added for binding with the lysate.  The lysate is loaded onto a spin column (DNA binds to silica gel membrane) & centrifugation is done with the flow through being removed.  Pre wash & wash is done using the respective solutions with the flow through discarded after centrifugation.  This removes trace amounts of salt and protein contaminants.  The DNA is eluted using an elution buffer & the eluate is stored.  Thus, these are the steps to isolating bacterial genomic DNA. Bacterial Genomic DNA Isolation Visualizing presence of Genomic DNA
  9. 9. Bacterial Plasmid DNA isolation  The harvested bacterial culture is resuspended using a resuspension solution for disturbing the pellet  Then, the lysis solution is added to lyse the cells & the neutralization solution is added leading to a cloudy solution.  Centrifugation is done & the supernatant lysate is added to the spin column (DNA binds with silica column in high salt concentration) with the sample being centrifuged again.  The flow through is discarded in the centrifugation processes.  Pre wash & wash is done using the respective solutions with the flow through discarded after centrifugation to remove contaminants.  The DNA is eluted using an elution buffer & the eluate is stored.  Thus, these are the steps to isolating bacterial plasmid DNA. Bacterial Plasmid DNA Isolation Visualizing presence of Plasmid DNA
  10. 10. SDS PAGE  SDS PAGE is a technique used to separate proteins by electrophoresis.  Apart from charge & size, molecular mass plays an important role in electrophoretic mobility.  Preparation- Milli-Q water, Acrylamide, Bisacrylamide, SDS, APS & TEMED.  Acrylamide acts as the gel medium for protein movement & Bisacrylamide helps in crosslinking acrylamide molecules.  APS acts as a catalyst for polymerization & is a free radical agent & TEMED enhances this effect.  SDS helps in denaturation of proteins & imparts negative charge to them.  Thus, SDS- PAGE helps us to separate proteins effectively & is used in techniques like western blotting as well. Visualization of results Gel Apparatus
  11. 11. Immunofluorescence  Immunofluorescence is a microscopic technique using a fluorescent microscope for microbiological samples.  The specificity of antibodies are used to target the antigen using a fluorescent dye to specific bio molecular targets which is visualized.  Blocking using the Blocking buffer is done as BSA attached to membrane with no target protein attached which eliminates false positives.  Then, incubation is done by adding the primary antibody & secondary antibody which is linked to DAPI & Alexa Fluor Stain.  This process is possible due to the constant (structure) & variable region (antigen acceptor) which allows various secondary antibodies to act on it.  Thus, we visualize the samples using a fluorescent microscope to obtain the images. DAPI Stain Alexa Fluor Stain
  12. 12. Western Blot  Western blot is an analytical technique used to detect specific proteins in a particular sample.  First, the process begins with SDS-PAGE which helps to separate the proteins by denaturation & electrophoretic movement.  For the proteins to be more accessible to antibody detection, we need to transfer the separated proteins to nitrocellulose or PVDF membrane.  By capillary action & electro blotting, this process occurs.  Blocking is done by adding 3-5% BSA & 0.1% Tween-20.  The diluted protein attaches to the membrane with no target protein attached which eliminates false positives during antibody addition.  Incubation is done by adding a primary & secondary antibody.  Visualization is doe by chemiluminescent detection. Radiographic visualiza Sample Results
  13. 13. Western Blot Procedure
  14. 14. Principles of ELISA  ELISA is a quantifying technique for detecting & measuring proteins, peptides, antigens & antibodies.  Adsorption of the unknown antigen to the assay plate allows the addition of the antibody which facilitates antigen- antibody interaction.  A detection enzyme is generally linked to the antibody. E.g. Horseradish peroxidase and Alkaline phosphatase.  Detection is done by assessing enzyme activity by adding a substrate to give a measurable product.  Thus, non-binding material are generally washed away making it a powerful tool. Some types of ELISA Apparatus
  15. 15. Acknowledgments  I would like to convey my thanks to VIT University for the opportunity & support which made this experience possible.  The faculty of School of Bio sciences & Technology were very helpful & I sincerely thank them for their guidance.  Special thanks to Dr. Karthikeyan, Dr. Rasool, Dr. Gothandam, Dr. Babu & Dr. Ramalingam for making this possible.  The PHD scholars & research fellows patiently accommodated & taught me the techniques & instilled passion and knowledge in me.  Special thanks to Mr. Pavan Kumar, Mr. Ganesan, Ms. Madhuri, Mr. Dinesh, Mr. Prasanth, Ms. Shanthini, Mrs. Ajitha, Mrs. Pooja, Ms. Indira, Mr. Chaitanya, Mr. Srinivasan & Mr. Manoj.

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