Is it an effective method to isolate DNA from Gram-negative bacteria with only the use of a boil and freeze-thaw protocol, without any chemical purification? Also, is there a media to maintain the bacterial cultures before DNA extraction, that doesn't affect PCR products?
Hi Natalia Mavromati
The boiling method:
1.Centrifuge some of the nutrient broth culture medium containing bacteria with half McFarland turbidity.
2. Discard the supernatant, then add 1 ml of Mili Q water to the precipitate.
3- Pour some suspension in sterile microtube and after closing it with parafilm, put it at 100 ° C for 15 minutes.
4.Centrifuge it for 1 minute and 5000 rpm.
5- Add 200% of 99% cold ethanol to 200 microliters of supernatant and put it in the freezer at -20 degrees Celsius for 1 hour.
6.Centrifuge it for 10 minutes at 13,000 rpm.
7- The supernatant was discarded and the microtubes were placed upside down on a paper towel under the laminar hood for 15 minutes to evaporate the ethanol.
8- Add 50 microliters of Mili Q water to each microtube and keep it in the freezer at -20 degrees Celsius.
Good luck
Dear Natalia
You can isolate DNA from bacteria using the "Isolation Kit"
You can grow bacteria onto normal NA plate
If isolation will be done using Kit, there will not be any problem in PCR amplification
Best wishes
Ajar Nath Yadav
You could also inoculate a single colony in 20 ul of nuclease free water and give an heat shock treatment for a minute and use 1 ul in 20 ul PCR reaction.
Most folks will make a streak plate with the colonies/strains they are investigating, grow them overnight, and wrap it really well in paradigm and store the plate in the refrigerator. As long as the agar doesn't dry out you can keep it for weeks or months.
Make sure you're using selective plates so you don't get plasmid drop-out or other issues.
You may use boiling method. I've done this for my previous project and it works. Based on my experience I didn't encounter any problem in PCR when I use the DNA extracted using boiling method without any purification. But I used the DNA to amplify 16s rDNA gene and few other housekeeping genes.
Thank you.
HI, Natalia Mavromati
Just as the others, just boil it, centrifuge and take the upper part without disturbing the pellet and it works fine enough.
Two extra tips: 1) I did not boil it (85-95ºC for 10-15 min is fine enough and do not break the DNA strands as much as If you do it at 100ºC);
2) After taking the sample from the centrifuge, do aliquot it asap and freeze it. Degradation occurs faster with boiling extraction ...
Wishing you all the best in your experiments.
Good vibes.
Hi Natalia
1. DNA extraction:
DNA Purification Kit Protocol - Promega
2. Prepare the bacterial cultures for DNA extraction
One loop-full of suspension from overnight growth bacterial isolates grown on TSA ( Tryptone Soy Agar ) were inoculated in test tube containing 5 ml sterilized TSB incubated overnight at 27-28 °C with shaking 120 rpm
Good Luck
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