Experiment-1

Aim: To extract DNA from the plant tissue.

Requirement

Theory: DNA extraction is the isolation of DNA from the tissue .

There are four basic steps in a DNA extraction:

1. Breaking the cells open, commonly referred to as cell disruption or cell lysis, to expose
the DNA within. This is commonly achieved by chemical and physical methods-
blending, grinding or sonicating the sample.
2. Removing membrane lipids by adding a detergent or surfactants.
3. Removing proteins by denaturing of degrading protein
4. Precipitating the DNA with an alcohol — usually ice-cold ethanol or isopropanol. Since
DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon
centrifugation. This step also removes alcohol-soluble salt.

All the steps we need some combination of buffers in which we can carry on the experiments.
Depending on the material and requirement the extraction process can be modified with
alteration of chemicals used.

Procedure:

1.100 mg of diseased and healthy leaf tissues were taken and washed with distilled water. After
drying, leaf tissues were ground in a pestle and mortar using liquid nitrogen.

2. The grounded powder was transferred to eppendorf tubes containing 1 ml of 950C preheated
extraction buffer and vortex vigorously.

3. The tubes were incubated at 950C for 10 min then kept on ice for 5 min.

4. The tubes were centrifuged at 12,000 rpm for 5 min.

5. Supernatant was transferred into new eppendorf tubes. To this tube 2µl of rnase A (100mg/ml)
was added and kept at 37 0C for 30 min.

6. To this 0.6 volume of (extract supernatant) ice cold isopropanol was added and kept on ice for
10 min.
7. Tubes were centrifuged at 12,000 rpm for 5 min.

8. The supernatant was discarded and 50 µl sterile distilled water was added to dissolve the
pellet.

9. To this 0.1 volume of 3M sodium acetate and 2 volumes of 95% ethanol were added and kept
at -20º C for 2 hrs.

10. Tubes were centrifuged at 12,000 rpm for 15mins.

11. Supernatant was discarded; to this 70% ethanol was added and centrifuged at 12,000 rpm for
2 min.

12. Again supernatant was discarded and the tubes were kept for 20 min at room temperature to
dry the pellet.

13. To the dried pellet 75 µl of sterile distilled water was added and the pellet was dissolved and
stored at -20 0 C for further use.

Result:

A translucent pellet was observed which dissolved in sterile distilled water.

Experiment No. 2

Objective: Amplification of isolated DNA.

Theory: PCR (Polymerase Chain Reaction) is a revolutionary method developed by Kary

Mullis in the 1980s. PCR is based on using the ability of DNA polymerase to synthesize new
strand of DNA complementary to the offered template strand. Because DNA polymerase can add
a nucleotide only onto a preexisting 3'-OH group, it needs a primer to which it can add the first
nucleotide. This requirement makes it possible to delineate a specific region of template
sequence that the researcher wants to amplify. At the end of the PCR reaction, the specific
sequence will be accumulated in billions of copies (amplicons).

Materials:

PCR machine
Micropipette
Water
Taq buffer
dNTP’s
MgCl2
Reverse Primer
Forward Primer
Taq polymerase
Template DNA

Procedure:

Preparing the PCR Reaction

Prepare the reaction mix using following components:

1. Allow the components to thaw on ice.

2. Prepare the reaction on ice according to given table:

Components Volume (µl)

Water 14 µl

Taq buffer 2.5 µl

dNTP’s 0.5 µl

MgCl2 1.5 µl

Reverse Primer 0.5 µl

Forward Primer 0.5 µl

Taq Polymerase 0.5 µl

Template DNA 5µl

 Total Volume 25 µl

3. Mix gently and place the reaction mix on ice

Performing Polymerase chain reaction:

4. Program the thermal cycler conditions using one of the thermal cyclers.

5. Set the reaction volume to 25μL.

6. Set the temperature and cycles:

The temperature regime followed for the PCR reaction was as below:
940 C for 5 min
940 C for 45 sec
550 C for 45 Sec
72 0 C for 1 min
72 0 C for 10 min
Cooling to 40 C
For 30 Cycles

7. Load the reactions into the thermal cycler.

8. Start the run.

Precautions:
Prepare the reaction mixture on ice.

Experiment No. 3
Aim: Small scale plasmid preparation from E. coli

Principle: Alkaline lysis plasmid miniprep is a procedure used to extract plasmid DNA from
bacterial cell suspensions. Plasmids are relatively small extrachromosomal supercoiled DNA
molecules while bacterial chromosomal DNA is much larger and less supercoiled. Therefore, the
difference in topology allows for selective precipitation of the chromosomal DNA, cellular
proteins from plasmids and also RNA molecules. Under alkaline conditions, both nucleic acids
and proteins denature. They are renatured when the solution is neutralized by the addition of
potassium acetate. Chromosomal DNA is precipitated out because the structure is too big to
renature correctly; hence plasmid DNA is extracted efficiently in the solution.

Requirements: Overnight grown bacterial culture, Sterile eppendorf tubes, Sterile microtips,
Micropipette, Plasmid Solution I, II and III, RNAse, Phenol: chloroform: isoamyl alcohol,
Isopropanol, Sodium acetate, Ethanol, TE buffer.

Procedure:
0
1. Grow 2 ml culture with appropriate antibiotic for 4-5 hrs at 37 C in a shaker till log
phase (Check for the turbidity of the culture)
2. Take 1.5 ml culture from each tube in an eppendorf tube (1.5 ml), spin at 10000 rpm for 2
min., remove the supernatant, spin down the rest of 3 ml culture in the same eppendorf
tube, 1.5 ml at a time. (Final culture spun, 4-5 ml)
3. Resuspend the cells in 100 μl of Solution I (Tris, EDTA, Glucose) (Suspend well by
vigorous vortexing)
4. Immediately add 200 μl of freshly prepared Solution II (0.4 N NaOH and 2% SDS, 1:1).
Mix by inverting the tube. Add 150 μl of Solution III (ice cold) to each tube, Mix by
inverting, Spin at 12,000 rpm for 15 min.
5. Transfer the supernatant to a fresh tube (carefully by avoiding the interphase), add 5 μl of
RNAse (10 mg/ml) to each individual tube, mix by inverting, give a pulse spin, incubate
0
at 37 C (water bath) for 1 hr
6. Add equal volume of phenol (200 μl) and then chloroform : isoamyl alcohol (200 μl),
mix by vortexing vigorously (Do not vortex vigorously for plant genomic DNA)
7. Spin at 12,000 rpm for 15 mins.
8. Transfer the supernatant carefully to fresh eppendorf, add equal volume (400 μl) of
Propan –2– ol and then 0.1 volume (40 μl) of Sodium acetate (pH 5.2) to each tube, mix
0
by inversion, keep in –20 C (Over night).
0
9. Spin at 12000 rpm, 4 C for 15 mins.
10. Discard the supernatant, add 200 μl of ice cold 70% ethanol, mix by inverting, spin at
0
12000 rpm, 4 C for 5 mins.
11. Discard the supernatant by using pipette, dry the pellet in laminar air flow.
12. Dissolve the pellet in 40 μl TE (10 mM Tris+1 mM EDTA), mix by tapping and give a
0
short spin. Store at –20 C.