1. Internship Progress Report
By Kailash Jayachandran
16INT0001

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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. Western Blot Procedure

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. 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.