DAY 01: Overview of Entomo-molecular Techniques

Experiment 01
Aim: Extraction of genomic DNA from adult mosquito sample.
Approach
1. Two adult mosquitoes were collected using mouth sucker and transferred to a clean test tube.
2. The opening of the test tube was sealed using a cotton plug and few drops of chloroform was added
using a pasteur pipette to anesthetize the mosquitoes.
3. The wings and appendages of the mosquitoes were carefully removed on a dissecting microscope
using forceps and needle.
4. The mosquito sample was then transferred to a 1.5 ml microcentrifuge tube (MCT) using a thin
liner brush.
5. 60 µl of lysis buffer 1 was added to the MCT and the mosquito sample was homogenized using a
micropestle.
6. The homogenized solution was incubated in a thermomixer at 70⁰C and 900 rpm for 25 mins.
7. 60 µl of lysis buffer 2 was further added to the sample and the solution was invert mix.
8. The solution was then incubated at room temperature (RT) for 5 mins.
9. The supernatant containing genomic DNA of mosquitoes was collected and all other debris within
the MCT was discarded.
10. The extracted genomic DNA (gDNA) was resuspended in 180 µl of nuclease free water to maintain
the stability of the gDNA. (Storage: 4⁰C for short term use and -20⁰C for long term use).

 Quantification of DNA: The concentration and purity of the extracted DNA can be measured using
NanoDrop spectrophotometers. This ensures the quality of the DNA for downstream applications.
Experiment 02
Aim: To amplify target DNA sequences using standard PCR.
Approach
1. The 2X PCR TaqMixture was thawed at room temperature.
2. The following reaction mix was prepared on ice:

Reagents Amount to be added

for each sample (µl)

Molecular Biology Grade Water 7

2X PCR TaqMixture 11

Forward Primer 1
(Concentration = 10 µM)

Reverse Primer 1
(Concentration = 10 µM)

Template DNA 2.5

3. The reaction mix was centrifuged in a microcentrifuge for 5 seconds.

4. The reaction mix was then placed in a thermal cycler and PCR was performed using standard
parameters as outlined below:

Steps Temperature (⁰C) Time Number

of Cycles

Initial Denaturation 94 5 mins 1

Denaturation 94 30 secs

Annealing 53 40 secs 30
Extension 72 1 min

Final Extension 72 5 mins 1

 Hold: The cyclic reaction can be programmed to end by holding the tubes at 10°C temperature for
several hours. In this cycle, the polymerase activity is minimized that might occur at higher
temperatures, although this is not usually a problem.
Experiment 03
Aim: To separate DNA (PCR products) by size (length in base pairs) using Agarose Gel Electrophoresis
for visualization and purification.
Approach

A. Preparation of 1.5 % Agarose gel

1. 1.5 g of agarose was measured on a weighing machine.
2. The agarose powder was then mixed in 100 ml of 1X TAE in a microwave conical flask.
(* Refer any protocol to prepare 10X TAE stock and then prepare 1X TAE working solution from the
stock).
3. The mixture was microwaved at Power:06 for 2-3 mins until the agarose was completely dissolved
(*But donot over boil the solution, as some of the buffer will evaporate and thus alter the final
percentage of agarose in gel).
4. The agarose solution was then allowed to cool down upto approximately 50°C (about when you can
confortably keep your hand on the flask) for 5 mins.
5. 4 µl of Ethidium Bromide (EtBr) was added to the 100 ml gel.
6. The agarose was then poured into a gel tray with the well comb in a place.
(* Pour slowly from one corner to avoid bubbles which will disrupt the gel. Any bubbles can be pushed
away from well comb or towards the side/edges of the gel with a pipette tip.
7. The gel was then left undisturbed at RT for 30-45 mins until it had completely solidified.

B. Loading samples and Running an Agarose Gel

1. After the gel was solidified it was placed into the gel box (electrophoresis unit).
2. The gel box was filled with 1X TAE until the gel is properly dipped.
3. 4 µl of 6X loading dye was added to each PCR product containing 20 µl sample and mixed well using
pipette.
4. 10 µl of sample was carefully loaded into each well of the gel.
5. 5 µl of a 1 kb molecular weight ladder was then carefully loaded into one of the well of gel.
6. The gel was run at 90-110 V until the dye line is approximately 75-80% of the way down the gel.
Typical run time was about 45 mins (depends on gel concentration and volatge).
(*Black is negative, red is positive. The DNA is negatively charged and will run towards the positive
electrode. Always Run towards Red).
7. Power was then turned off, the electrodes were disconnected from the power source, and the gel was
carefully removed from the gel box.
8. The DNA fragments were visualized under UV Transilluminator.
(* The fragments of DNA referred as bands, due to their appearance on gel)
Inference for Experiment 01, 02, and 03
1. When the gel was exposed to a short wave UV light source, electrons in the aromatic ring of the EtBr
were activated that led to the release of energy (light) as the electron returned to the ground state. The
phosphate backbone of DNA molecule is negatively charged, therefore when placed in an electric
field, DNA fragments migrated to the positively charged anode.
2. Potential by-product in PCR reaction called primer-dimer was obtained. These primer dimers cosnists
of primer molecules that have attached, hybridized to each other because of strings of complementary
bases in the primers and confirms that the reaction contained all components necessary for
amplification.
3. 1.5 % agarose gel electrophoresis image represents molecular weight ladder and the bands of PCR
products at the target sequence 700 bp (with reference to the molecular ladder).

 Attach your gel image and level the molecular ladder and bands observed.
Experiment 04
Aim: To learn the technique of DNA purification from agarose gel using column based method.
Approach
1. DNA bands were excised from the EtBr stained gel using a clean razor blade or scalpel blade under
312 nm UV light and stored it in a pre-weighed plastic micro centrifuge tube at 4° C.
(* Cut above and below each fragment of interest minimizing the amount of agarose in the slice. Also
minimize the amount of time the DNA is exposed to UV by having sterile pre-weighed labeled tubes
ready for the slices to avoid degradation of DNA).
 Experiment not performed. The below content is provided just for future reference.
2. Weigh the gel slice and accordingly add 3 volumes of Gel Bind Buffer per gel slice volume. Incubate
at 55° C for 10 minutes with intermittent mixing every 2-3 minutes so that the agarose dissolves
completely (the yellow colour of Gel Bind Buffer signifies a pH of < 7.5).
(*For e.g. 100 mg of agarose gel slice requires 300 μl of Gel Bind Buffer).
NOTE: Monitor the pH of the Gel: Gel Binding Buffer mixture after the gel has completely dissolved.
If the color of the mixture turns violet, add 5 µl of 5M Sodium acetate (pH 5.2) to bring down the pH.
After this adjustment, the color of the Gel: Gel Binding Buffer mixture should be light yellow. The
agarose gel slice should be solubilized completely.
3. Load sample onto the column.
Load the sample onto the HiElute Miniprep Spin column, and centrifuge at 13,000 rpm for 1 minute.
NOTE: The maximum binding capacity of the column is 700 μl. For sample volumes exceeding 700
μl repeat the column step for the remaining sample.
4. Discard the flow through and place the HiElute Miniprep Spin column in a new 2.0 ml collection tube.
5. Add 300 μl of Gel Bind Buffer (HG) into the column and centrifuge for 1 minute at 10,000 x g
(≈12,000 rpm) at room temperature (15-25°C) to wash the membrane. Discard the flow-through and
reuse the collection tube.
6. Wash
Add 750 μl of the Gel Wash Buffer to the HiElute Miniprep Spin column placed in a new 2.0
collection tube and centrifuge for 1 minute at 13,000 rpm.
7. Discard the flow through and place the HiElute Miniprep Spin column in the same collection tube.
Spin for an additional 1 minute at 13,000 rpm.
8. Carefully transfer the HiElute Miniprep Spin column to a clean 2.0 ml collection tube provided.
9. Elution
Add 50 μl of Elution Buffer directly onto the center of the HiElute Miniprep Spin column. Incubate at
room temperature (15-25°C) for 1 minute.
NOTE: Incubation at room temperature (15-25°C) for 1 minute increases the elution efficiency.
10. Centrifuge at 13,000 rpm for 1 minute to elute DNA.
NOTE: All centrifugation steps are carried at room temperature (15-25°C). For short-term storage
 (24-48 hours) of the DNA, 2-8°C is recommended. For long-term storage, -20°C or lower
temperature (-80°C) is recommended. Avoid repeated freezing and thawing of the sample which
may cause denaturing of DNA. The Elution Buffer will help to stabilize the DNA at these
temperatures.
11. Quantitate the concentration of the DNA using a spectrophotometer or estimate the concentration by
comparing its intensity with that of a DNA ladder of known concentration.