Msc biotechnology 2nd sem all experiments notes for e.g. BLASTA ,FASTA,SDS PAGE ,agarose gel electrophoresis, etc.

1. KAUSHIK
16
BIOTECHNOGY
PRACTICAL
SEM 2.
ABHISHEK KAUSHIK

2. Experiment No. 1
Aim: To isolate the genomic DNA from E .coli.
Principle:-The isolation of DNA from bacteria is a relatively simple process. The organism to be used should
be grown in a favorable medium at an optimal temperature, and should be harvested in late log to early stationary
phase for maximum yield.The genomic DNA isolation needs to separate total DNA from RNA,protein, lipid, etc.
Initially the cell membranes must be disrupted in order to release the DNA in the extraction buffer. SDS (sodium
dodecyl sulphate) is used to disrupt the cell membrane
Materials Required:
􀂾 LB Broth
􀂾 E. coli DH5α cells
􀂾 Reagents
􀂾 TE buffer (pH 8.0)
􀂾 10% SDS
􀂾 Proteinase K
􀂾 Phenol-chloroform mixture
􀂾 5M Sodium Acetate (pH 5.2)
􀂾 Isopropanol
􀂾 70% ethanol
􀂾 Autoclaved Distilled Water
􀂾 Eppendorf tubes 2 ml
􀂾 Micropipette
􀂾 Microtips
􀂾 Microfuge
PROCEDURE:
􀂾 2 ml overnight culture is taken and the cells are harvested by centrifugation for 10 minutes
􀂾 875 μl of TE buffer is added to the cell pellet and the cells are resuspended in the buffer by gentle
mixing.
􀂾 100 μl of 10% SDS and 5 μl of Proteinase K are added to the cells.
􀂾 The above mixture is mixed well and incubated at 37º C for an hour in an incubator.
􀂾 1 ml of phenol-chloroform mixture is added to the contents, mixed well by inverting and incubated at
room temperature for 5 minutes.
􀂾 The contents are centrifuged at 10,000 rpm for 10 minutes at 4º C.
􀂾 The highly viscous jelly like supernatant is collected using cut tips and is transferred to a fresh tube.
􀂾 The process is repeated once again with phenol-chloroform mixture and the supernatant is
collected in a fresh tube.
􀂾 100 μl of 5M sodium acetate is added to the contents and is mixed gently.
􀂾 2 ml of isopropanol is added and mixed gently by inversion till white strands of DNA precipitates
out.
􀂾 The contents are centrifuged at 5,000 rpm for 10 minutes.
􀂾 The supernatant is removed and 1ml 70% ethanol is added.
􀂾 The above contents are centrifuged at 5,000 rpm for 10 minutes.
􀂾 After air drying for 5 minutes 200 μl of TE buffer or distilled water is added.
􀂾 10 μl of DNA sample is taken and is diluted to 1 or 2 ml with distilled water.
􀂾 The concentration of DNA is determined using a spectrophotometer at 260/280 nm.
􀂾 The remaining samples are stored for further experiments.
PRECAUTIONS:
􀂾 Cut tips should be used so that the DNA is not subjected to mechanical disruption.
􀂾 Depending on the source of DNA the incubation period of ProteinaseK should extended.
􀂾 The phenol chloroform extraction should be repeated depending on the source of DNA to obtain pure DNA.
􀂾 DNase free plastic wares and reagents should be used..
Caution:
Phenol causes severe burns. Wear gloves, goggles, and a lab coat,and keep tubes capped tightly.

3. EXERCISE 2
AIM:- TO ISOLATE THE GENOMIC DNA FROM PLANT SOURCE
Principle
The major given protocol describes a rapid method for the isolation of plant DNA without the use of
ultracentrifugation. The DNA produced is of moderately high molecular weight, which is suitable for
most restriction-end nucleases and genomic blot analysis (Delta portal, et al, 1983). This method also
helps to extract DNA from small amounts of tissues and a large number of plant samples, and is very
rapid.
Materials
*Plant tissue
*Young leaves and callus of hibiscus
*Pestle and mortar
*Icebox and ice
DNA extraction buffer
5M potassium acetate
3M sodium acetate buffer
Phenol choloroform
Isoamyl alcohol
100% ethanol (ice cold) 70% ice cold ethanol
Sterile eppendorf
Microcentrifuge and glass powder
Procedure
1. Use 500 mg of young plant tissue (stem/leaf/leaf callus of controlled or transformed plant), in a
precoated mortar and pestle.

4. 2. Grind it up inside the icebox with ice, using 0.8 mL of extraction buffer and glass powder.
3. Add the rest of the buffer. Pour the ground extraction into a test tube.
4. Add 0.2 mL of 20% SDS, shake, and inoculate at 65°C–75°C for 10 to 15 minutes.
5. Add 1.5 mL of potassium acetate (5 m).
6. Shake vigorously, and keep on ice for 20 minutes.
Result
The DNA from plant cells has become extranded and clear bands are seen when the DNA was seen
on the gel.
Experiment No. 3
Plasmid DNA Isolation
Aim: To isolate plasmid DNA from bacterial cells.
Principle:
Plasmids are extrachromosomal double-stranded closed-circular DNA present in many
microorganisms In nature, plasmids are large molecules with sizes ranging from 2 Kb to 100 Kb.
Plasmids have origin of replication and multiply by utilizing the long leaved enzymes of the host DNA
polymerizes.. Plasmid DNA needs to be extracted (from bacterial hosts, mostly E.coli) almost
routinely in cloning experiments.
Materials Required:
􀂾 Luria Broth
􀂾 Bacterial cells containing plasmid
􀂾 Reagents
􀂾 TE buffer(pH 8.0)
􀂾 Solution I (50 mM glucose, 25 mM Tris, 10 mM EOT A, pH 8.0)
􀂾 Solution II (0.2 M NaOH, 1% SDS)
􀂾 Solution III (5M Potassium acetate, 60 mL; acetic acid, 11.5 mL; distilled water, 28.5 mL).
􀂾 Phenol-chloroform mixture
􀂾 Isopropanol
􀂾 70% ethanol
􀂾 Autoclaved Distilled Water
􀂾 Eppendorf tubes 2 ml
􀂾 Micropipette
􀂾 Microtips
􀂾 Microfuge
Procedure:
1. Inoculate 2 ml of rich medium (LB, YT, or Terrific Broth) containing the appropriate antibiotic with a single colony
of transformed bacteria. Incubate the culture overnight at 37°C with vigorous shaking.
2. Pour 1.5 ml of the culture into a microfuge tube. Centrifuge at maximum speed for 30 seconds at 4°C in a
microfuge. Store the unused portion of the original culture at 4°C.
3. Remove the medium by aspiration, leaving the bacterial pellet as dry as possible.
4. Resuspend the bacterial pellet in 100 μl of ice-cold Alkaline lysis solution I by vigorous vortexing.
5. Add 200 μl of freshly prepared Alkaline lysis solution II to each bacterial suspension. Close the tube tightly, and
mix the contents well by inverting the tube . Do not vortex! Store the tube in ice.
6. Add 150 μl of ice-cold Alkaline lysis solution III. Close the tube and disperse Alkaline lysis solution III through the
viscous bacterial lysate by inverting the tube several times. Store the tube in ice for 3-5 minutes.
7. Centrifuge the bacterial lysate for 5 minutes at maximum speed at 4°C in a microfuge. Collect the supernatant to
a fresh tube.

5. 8. (Optional) Add equal volume of phenol: chloroform. Mix the organic and aqueous phases by vortexing and then
centrifuge the emulsion at maximum speed for 2 minutes at 4°C in a microfuge. Transfer the aqueous upper layer
to a fresh tube.
9. Precipitate nucleic acids from the supernatant by adding 2 volumes of ethanol at room temperature. Mix the
solution by vortexing and then allow the mixture to stand for 2 minutes at room temperature.
10. Collect the precipitated nucleic acids by centrifugation at maximum speed for 5 minutes at 4°C in a microfuge.
11. Discard the supernatant by aspiration. Stand the tube in an inverted position on a paper towel to allow all of the
fluid to drain away. Use a Kim wipe or disposable pipette tip to remove any drops of fluid adhering to the walls of
the tube.
12. Add 1 ml of 70% ethanol to the pellet and invert the closed tube several times. Recover the DNA by
centrifugation at maximum speed for 2 minutes at 4°C in a microfuge.
13. Remove all of the supernatant by aspiration. Take care with this step, as the pellet sometimes does not adhere
tightly to the tube.
14. Remove any beads of ethanol from the tube. Store the open tube at room temperature until the ethanol has
evaporated and no fluid is visible in the tube (5-10 minutes).
Dissolve the nucleic acids in 50 μl of TE (pH 8.0) containing 20 μg/ml DNase-free RNase A (pancreatic
RNase). Vortex the solution gently for a few seconds and store the DNA at -20°C.
Result:- Detect the plasmid by doing agarose gel electrophoresis
Experiment No. 4
Aim: To separate and visualise DNA bands by Agarose gel electrophoresis.
Principle:
The agarose gel contains molecule sized pores, acting like molecular sieves.The pores in the gel
control the speed that molecules can move. DNA molecules possess a negative charge in their
backbone structure due to the presence of PO4 -groups thus this principle is exploited for its
separation. Agarose gels are made with between 0.7% (provides good resolution of large 5–10 kb
DNA fragments) and 2% (good resolution for small 0.2–1 kb fragments). The gel setup provides wells
for loading DNA in to it. The loaded DNA molecules move towards the positively charged electrode
(anode) and get separated along the length of the gel. Ethidium bromide (EtBr), a chromogen is
added to the gel to visualize the separated DNA under UV transillumination.
Materials Required:
• Electrophoresis buffer: 1x TAE buffer
• Agarose ultra pure (DNA graded)
• electrophoresis tank, gel tray, sample comb and power supply
• Plastic or insulation tape
• Ethidium bromide: 10 mg /ml stock solution
• 5x Gel loading dye
• DNA marker solution, DNA sample and gloves.

6. PROCEDURE:
1. Making a 1% Agarose Gel
• Weigh 0.5 g agarose and dissolve it in 50 mL of 1x TAE Buffer.
• Heat the solution over a hot plate to boiling constituency marked with a clear solution
• Leave the solution to cool and add 2μl of EtBr solution mix it well by gentle swirling.
• Pour it in the gel tray-comb set up. Also be sure the gel plates have been taped securely and contain
the well combs prior to pouring
• Allow the solution to cool and harden to form gel.
2. Loading of Samples
• Carefully transfer the gel to the electrophoresis tank filled with 1x TAE buffer.
• Prepare your samples [8 ul of DNA sample (0. 1 ug to 1 ug) and 2 ul of 5x gel loading dye]
• Remove the comb and load the samples into the well.
• Connect appropriate electrodes to the power pack and run it at 50-100volts for 20min.
• Monitor the progress of the gel with reference to tracking dye (Bromophenol blue). Stop the run
when the marker has run 3/4 th of the gel.
3. Examining the gel
• Place the gel on the UV-transilluminator and check for orange colored bands in the gel.
PRECAUTIONS:
􀂾 Wear gloves during the addition of EtBr and while handling the casted gel (EtBr is a potent
carcinogen).
􀂾 Handling the gel should be careful as the gel may break due to improper handling.
RESULT
the UV-trans illumination for visualising the bands, avoid direct contact and exposure to eyes.
Experiment No. 5
AIM:- To Pairwise Sequence Alignment using BLAST
PRINCIPLE-
BLAST program was designed by Stephen Altschul, Warren Gish, Webb Miller, Eugene Myers, and
David J. Lipmann at National Institutes of Health (NIH) and was published in Journal of Molecular
Biology in 1990. BLAST (Basic local alignment search tool) is a heuristic search algorithm, it finds the
solutions from the all possibilities ,which takes input as nucleotide or protein sequence and compare it
with existing databases like NCBI, GenBank etc. It finds the local similarity between different
sequences and calculates the statistical significance of matches. It can also be used to find functional
and evolutionary relationship between different sequences.
Procedure :-
Step 1: Select the BLAST program
User have to specify the type of BLAST programs from the database like BLASTp, BLASTn, BLASTx, tBLASTn,
tBLASTx.
Step 2: Enter a query sequence or upload a file containing sequence

7. Enter a query sequence by pasting the sequence in the query box or uploading a FASTA file which is having the
sequence for similarity search. This step is similar for all BLAST programs. The user can give the accession
number or gi number or even a raw FASTA sequence. Go to simulator tab to know more about how to retrieve
query sequence.
Step 3: Select database to search
User first has to know what all databases are available and what type of sequences are present in those
databases. Sequence similarity search involves searching of similar sequences of the query sequence from the
selected databases
Step 4: Select the algorithm and the parameters of the algorithm for the search
There are different algorithms for some of the BLAST program. User has to specify the algorithm for the BLAST
program. Nucleotide BLAST uses algorithms like MegaBLAST which searches for highly similar sequences,
discontiguous MegaBLAST which searches for more dissimilar sequences and BLASTn which searches for
somewhat similar sequences. Meanwhile for protein BLAST algorithms like BLASTp, searches for similarity
between protein query and protein database, PSI-BLAST performs position specific search iteratively, PHI-
BLAST searches for a particular pattern (user has to enter the pattern to search in the PHI pattern box provided)
that is present in the sequence against the sequences in the database, DELTA-BLAST is Domain Enhanced
Lookup Time Accelerated BLAST. It searches multiple sequence and aligns them to find protein homology.
Step 5: Run the BLAST program
Submission of the BLAST program can be done by clicking the BLAST button at the end of the page. Screen
shot of result can be shown in
BLAST Result:
After submitting the query sequence for sequence similarity search, the result page will appear along with the
information like Query id, Description, Molecule type, Length of sequence, Database name and BLAST program.
It shows the putative conserved domains that have been detected while undergoing sequence similarity search.
Finally it shows the actual alignment, along with the query sequence on the top and database sequence below
the query. The number on either sides of the alignment indicates the position of amino acids/nucleotides in
sequence which can be represented.
fig. No. 1
Fig. No 2

8. Fig. No. 3
Fig. No. 4
EXPRIMENT NO. 6
AIM:- To Pairwise sequence alignment using FASTA
Principle:-
FASTA is a pairwise sequence alignment tool which takes input as nucleotide or protein sequences and
compares it with existing databases It is a text-based format and can be read and written with the help of text
editor or word processor. Fasta file description starts with ‘>’ symbol and followed by the gi and accession
number and then the description, all in a single line. Next line starts with the sequence and in each row there
would be 60 nucleotides/amino acids only. FASTA program uses the word hits to identify potential matches
before attempting the more time consuming optimised search. The speed and sensitivity is controlled by the
parameter called ktup, which specifies the size of the word.
Procedure:-
Step 1: Specify the tool input
Select the database to search :Databases are required to run the sequence similarity search. Multiple
databases can be used at the same time. The different databases are
Uniprot Knoweldge base
Uniprot KB/swiss-prot
Uniprot KB/ Swissprot isoforms
Uniprot KB /Trembl
UniProtKB Taxonomic Subsets

9. UniProt Clusters
Patents
Structure
Step 2 Entering of input sequence
The query sequence can be entered directly in GCG, FASTA, EMBL, GenBank, PIR, NBRF, PHYLIP or
UniProtKB/Swiss-Prot formats.
Sequence file upload
A file containing the valid sequence in any format mentioned above can be used as a query for sequence
similarity search. Sequence type indicates the type of sequence (PROTEIN / DNA / RNA) for similarity search.Go
to simulator tab to know more about how to retrieve the query sequence.
Step 3: Setting up parameters
User has to specify the type of program and the matrix for scoring. FASTA, FASTX, FASTY, SSEARCH,
GGSEARCH and GLSEARCH are the different programs used. Substitution matrix are used for scoring
alignments. The matrices are BLOSUM50, BLOSUM62, BLASTP62, PAM120, PAM250, MDM10, MDM20, and
MDM40. BLOSUM50 is set as a default substitution matrix. Parameters include.
Step 4: Submission
The result page can be seen in another window by clicking submit. This is an interactive process, when the
process is complete the result will be displayed in the browser. Result can be sent to a valid email address which
has to be specified in the text box.
FASTA Result Analysis
Summary Table :-Result page appears by giving the information like aligned sequences from the
sequence similarity search, database id, source of the sequence, Gene-expression, molecule type,
Nucleotide sequence, Genomics, Protein sequences, Ontologies, Enzymes, protein families, and
Literature, which is followed by the length of sequence, score, identities, positives and E-value.

10. EXPERIMENT NO. 7
AIM:- Aligning Multiple Sequences with CLUSTAL W
PRINCIPLE:-
Sequence is a collection of nucleotides or amino acid residues which are connected with each other.
Speaking biologically, a typical DNA/RNA sequence consist of nucleotides while a protein sequence
consist of amino acids. Sequencing is the process to determine the nucleotide or amino acid sequence
of a DNA fragment or a protein. There are different experimental methods for sequencing, and the
obtained sequence is submitted to different databases like NCBI, Genbank etc.
PROCEDURE:-
1.To download the data , and to get acces to the tools, go to simulator tab.
2.Get access to the CLUSTALW tool.
3. In the dialog box given, paste your set of sequences, the sequences should be pasted with the ‘>’ symbol followed
by name of the sequence (as similar as FASTA format) followed by return (enter key) and then the sequence
4. The sequences can also be submitted through file by clicking on the option “choose file” such that all the
sequences should be in similar format.
5. The other two steps the user can select on his/her own to set the parameters for pair wise alignment options
and multiple sequence alignment options, to select the scoring matrices and scoring values.
6. After the submission of the job the results can be downloaded into a file by clicking on the option Download
alignment file.
RESULTS

11. The result files are with different formats of input and output files of the alignment.The user can enable the java
plug-in in the browser, if it is disabled and thus the user can use Jalview to see the alignment. The output file
represents the length of each sequence , and the score of each alignment individually.

12. EXPERIMENT NO. 8
AIM:- TO Phylogenetic Analysis using PHYLIP - Rooted trees
PRINCIPLE:-
PHYLIP is a complete phylogenetic analysis package which was developed by Joseph Felsestein at University of
Washington. PHYLIP is used to find the evolutionary relationships between different organisms. Some of the
methods available in this package are maximum parsimony method, distance matrix and likelihood methods. The
data is presented to the program from a text file, which is prepared by the user using common text editors such
as word processor, etc.
Procedure:-
1.Align the multiple DNA sequences (output of the ClustalW) and save it in PHYLIP format as infile.phy. Start the
program of Dnadist by clicking the icon and giving this infile as input.
2. All the PHYLIP programs are menu driven programs. Dnadist will calculate pairwise distances between the
sequences
3. At first, Dnadist will ask whether the input file is there in the PHYLIP folder. If the file does not exist, it will ask
you to give the correct file name.
4. After giving the correct input, if needed it will ask to change any settings for the program by typing the first
letter or number. If the changes are not required, by typing ‘Y’ it will start running the program. Output will return
to the file as outfile, so that the output of this file can be used as input of another program.
5. Like Dnadist, Neighbor also gives sequence distance analysis. Output of Dnadist is given as input to Neighbor.
Output file and tree file will be returned to outfile and outtree. Branch lengths and tree are represented with the
help of Neighbor joining method.
RESULTS:-
Rooted trees are represented via Drawgram by providing the input as the "outfile" obtaining from neighbor joining
method. Rooted tree considers an imaginary root as the start and from that the other sequences are aligned.
EXPERIMENT 9

13. AIM:- To SEPARATION OF PROTEIN STANDARDS: SDS-PAGE
PRINCIPLE:-
SDS PAGE stands for sodium dodecyl sulphate (SDS) POLYACARYLAMIDE gel electrophoresis(PAGE) and is a
method useful in separation and identification of proteins according to their size. Since different proteins with
similar molecular weights may migrate differently due to their differences in secondary, tertiary or quaternary
structure, SDS which is an anionic detergent is used in SDS PAGE to reduce proteins to their primary structure
by breaking up the complex structure into their polypeptide subunits in the process of denaturation of protein. it
then coats them with uniform negative charge by the process called denaturation.
Materials requered
10% SDS-polyacrylamide gel
Protein standards
2X-SDS sample buffer
1X-SDS electrophoresis running buffer (Tris-Glycine + SDS)
0.001% (w/v) bromophenol blue
Micropipettes with flat tips for electrophoresis wells
Procedure
1. Remove the Teflon combs from the prepared gels by gently lifting the combs from the chamber. Rinse the
wells (formed by the removal of the combs) with distilled water and drain it off.
2. Fill the wells and the chamber with running buffer.
3. Prepare aliquots of a known protein standard by mixing equal parts of the protein standard with 2X sample
buffer.
4. Using a micropipette, add the sample to the bottom of a well. Add the blue to a separate well.
5. Remove the gel from its casting stand and assemble it into the appropriate slab unit for running the
electrophoresis. Be sure to follow the manufacturer’s directions for assembly.
6. Pour a sufficient quantity of running buffer into both the lower and upper chambers of the electrophoresis
apparatus until the bottom of the gel is immersed in buffer, and the top is covered, while the electrodes reach
into the buffer of the upper chamber. Be careful not to disturb the samples in the wells when adding buffer to the
upper chamber.
7. Assemble the top of the electrophoresis apparatus and connect the system to an appropriate power source. Be
sure that the cathode (+) is connected to the upper buffer chamber.
8. Turn on the power supply and run the gel at 20 mA constant current per 1.5 mm of gel.
9. When the tracking dye reaches the separating gel layer, increase the current to 30 mA per 1.5-mm gel.
10. Continue applying the current until the tracking dye reaches the bottom of
the separating gel layer (approximately 4 hours).
11. Turn off and disconnect the power supply. Disassemble the gel apparatus and remove the glass sandwich
containing the gel. Place the sandwich flat on paper towels and carefully remove the clamps from the sandwich.
12. Working on one side of the sandwich, carefully slide 1 of the spacers out from between the 2 glass plates.
Using the spacer or a plastic wedge as a lever, gently pry the glass plates apart without damaging the gel
contained within.
13. Lift the bottom glass plate with the gel and transfer the gel to an appropriate container filled with buffer,
stain, or preservative. The gel may at this point be used for Coomasie Blue staining, silver
staining, enzyme detection, Western blots, or more advanced procedures, such as electroblotting or
electroelution.
14. Plot the relative mobility of each protein against the log of its molecular
weight.
Relative mobility is the term used for the ratio of the distance the protein has moved from its point of origin (the
beginning of the separating gel) relative to the distance the tracking dye has moved (the gel front). The ratio is
abbreviated as Rf. Molecular weight is expressed in daltons, and presents a plot of the relative molecular weight
of protein standards against the log of their molecular weight.
RESULT

14. Abhishek kaushik