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Genomic Dna Isolation Protocol From Bacteria

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Md Bulbul Ahmed
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26 views

Genomic Dna Isolation Protocol From Bacteria

Uploaded by

Md Bulbul Ahmed
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd
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Genomic DNA isolation from Bacillus thuringiensis

There are many methods to isolate genomic DNA from bacteria such as enzymatic lysis, thermal
shock, mechanical lysis method. Here a simple method of Genomic DNA isolation from Bacillus
thuringiensis based on thermal shock is described as per Bravo et al. (1998).

Materials required:

1) Preserved bacterial isolates


2) Nutrient Agar media
3) Inoculation loop
4) Petri-dishes
5) Spirit lamp and Laminar Hood
6) Micro-pipette
7) Micro tube (1.5ml)
8) Micro-pipette tips
9) Sterile double distilled water
10)

Genomic DNA isolation protocol:

1. Before inoculating bacteria, prepare petri dishes with sterile Nutrient Agar (~20ml/plate).
2. Open the tube from the preserved Bt stocks in glycerol, and with the help of a sterile loop
or toothpick, scrape some of the frozen bacteria on the top. Do not let the glycerol stock
unthaw!
3. Streak the bacteria onto a nutrient agar plate on a sterile condition through inoculation
loop under the Laminar Hood with the aid of Spirit lamp.
4. Cover the plate with scotch tape or
5. Incubate agar plates at the 25-27°C temperature for 24 hours to grow the bacterial
colonies.
6. Transfer 100 μl (0.1 ml) of sterile distilled H2O in a 1.5ml tube by Micro-pipette.
7. Sterile the inoculation loop on the spirit lamp and cool down.
8. Pick a Bt single colony with an inoculation loop from the plate and transfer to above
1.5ml tube containing 100 μl (0.1 ml) of sterilized distilled H 2O and mixed thoroughly
with the help of the inoculation loop.

The inoculation loop must be sterile under spirit lamp each time for each Bt isolate
after transfer. High precautions needed so that different Bt isolates are NOT mixed
and contaminated.

9. Keep the mixture (100 μl) at −20°C freezer for about 1-2 hours.
10. Prepare the incubator set to boiling temperature (100°C).

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11. Transfer the mixture from freezer to incubator at boiling water (100°C) and keep for
minimum 10 min to lyse the cells.
12. Prepare the centrifuge machine.
13. Centrifuge the resulting cell lysates briefly at 10,000 rpm for 10-30s.
14. After being centrifuged, take about 80-90μl of supernatant which will be used as DNA
sample in the PCR. If debris come with the micropipette tip, then a second centrifuge is
necessary.
15. Determine the concentration of the DNA by gel electrophoresis or nano-spectrometer.
16. For PCR, about 5–50 ng of gDNA may be required as a starting amount in a 50 µL PCR
volume. For this reason, take about 5-15μl of supernatant may be required as DNA
sample in the PCR reaction mixture.
17. Prepare several aliquots of DNA material (20-50 μl) and store at -20°C. Use only one
aliquot at a time. If more amount of DNA is require, then several (3-5) extraction should
be done.
18. DNA stored long term should be in ultra-low freezers, typically at or below -80°C which
should prevent the degradation of nucleic acids in the DNA.

Reference:

1. Bravo, A., Sarabia, S., Lopez, L., Ontiveros, H., Abarca, C., Ortiz, A., Ortiz, M., Lina, L.,
Villalobos, F. J., Peña, G., Nuñez-Valdez, M. E., Soberón, M., & Quintero, R. (1998).
Characterization of cry genes in a Mexican Bacillus thuringiensis strain
collection. Applied and environmental microbiology, 64(12), 4965–4972.
https://doi.org/10.1128/AEM.64.12.4965-4972.1998

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REAGENTS NEEDED FOR PCR Protocol

1. Taq DNA polymerase


2. PCR grade water PCR Reagent Water
3. Primers diluted to working concentration: 10 µM working stocks are sufficient for most
assays.
4. DNA template
5. Dedicated pipettes
6. Thermal cycler with various well block sizes or in multi format
7. Sterile filter pipette tips
8. Sterile 1.5 mL screw-top micro centrifuge tubes with screw cap
9. PCR tubes or plates: Individual thin-walled 200 µL PCR tubes; 200 µL strip tubes;
Multiwell plates and plate seal
10. dNTP mix Deoxynucleotide mix containing 10 mM each of dATP, dCTP, dGTP, and
dTTP

PCR Protocol for confirmation of Bt by XRE gene:

1. Thaw all reagents on ice. The final volume should be 20 µL.


2. Add the reagents to an appropriately sized tube.
3. Add reagents in following order: water, buffer, dNTPs, MgCl 2, template primers, Taq
polymerase.
4. For a large number of reactions, a mastermix without the template should be set up and
aliquoted into reaction tubes. At the end, template should be added to appropriate tubes.
5. Mix gently by vortex and briefly centrifuge to collect all components to the bottom of the
tube. Add 50 µL of mineral oil to the top of each tube to prevent evaporation if using a
thermal cycler without a heated lid.
6. Then Amplify. The amplification parameters will vary depending on the primers and the
thermal cycler used. It may be necessary to optimize the system for individual primers,
template, and thermal cycler.
7. Program your thermocycler for your PCR reaction using the following guidelines:

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Typical Cycling Parameters

Sl. Step Temp Time No. of cycles


1. Initial Denaturation 94°C 5 min
2. Denaturation 94°C 30 sec
3. Primer Annealing Tm-5°C 45 sec 30-35
4. Extension 72°C 1 min per kb
5. Final Extension 72°C 5 min

8. Evaluate amplified DNA by agarose gel electrophoresis followed by ethidium bromide


staining.

Reagents for Nucleic Acid Electrophoresis

1. Agarose (precast gels, powder, etc.)


2. Buffer such as MOPS-EDTA-sodium acetate, tris-acetate-EDTA (TAE) or tris-borate-
EDTA (TBE)
3. Gel loading solution and sample loading buffer for RNA
4. Electrophoresis stain or dye such as ethidium bromide

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