If I want to extract the genomic DNA from bacteria, is the use of heating or boiling of bacteria an efficient method or should I try another method?
The aim of extraction is PCR and sequencing/
Hi,
For Screening PCR there is no harm using one ul o/n culture in PCR mix but remember for sequencing you would need a high quality product. Also primer binding will be dependent on salt concentration and thus using culture directly may not give reproducible results. Like suggested by Artur you should opt for convenient protocol from the list which is availed in maniatis or current protocols in molecular biology and some specific species specific references. Also using one ul would be fine for some Gram negative bacteria but not for Staphyloccus aureus and bacteria with tough cell wall, merely heating wont give quality DNA.
Thanks for your answer
I know that there are a lot of protocols of manual DNA extraction
But some friends had informed me that I can extract DNA by simpler method (by boiling or heating) and I want to make sure of it
Boiling method can be used for plasmid DNA isolation - also bacterial :-)
If this is to perform some PCR to screen, you can add 1ul of your overnight culture directly in the PCR mix!
Hi,
For Screening PCR there is no harm using one ul o/n culture in PCR mix but remember for sequencing you would need a high quality product. Also primer binding will be dependent on salt concentration and thus using culture directly may not give reproducible results. Like suggested by Artur you should opt for convenient protocol from the list which is availed in maniatis or current protocols in molecular biology and some specific species specific references. Also using one ul would be fine for some Gram negative bacteria but not for Staphyloccus aureus and bacteria with tough cell wall, merely heating wont give quality DNA.
Hey,
I've been using this kind of DNA extraction method for pure culture PCR.
Briefly, for 1mL sample: 10 min at 100°C, then 10 min at -20°C and then centrifugation for 5 min at max speed (14.000 rpm). The DNA remained in the supernatant.
To get rid of the salts from the culture media you can previously centrifuge the sample at max speed and add sterile water instead.
However, i completely agree with the comment before that for sequencing you will need a high quality product. You can check that by using a Nanodrop or a similar device.
You can also get a more purified DNA solution if you simply harvest your cell, resuspend them in 200 uL of water add 200 uL of chloroform centrifuged and get the supernantant. Enought for several PCRs even from gram possitive bacteria. But as mentioned before for sequencing you would need a more pure and concentrated DNA solution.
Yes, this method is fine. I do it all the time to check whether my cloning of cDNA into the bacterial vector was successful. PCR and sequencing works without a hitch.
Hi
Pellet 1ml culture, then add 100uL of sterile milliQ. Then boil it for 5 min at 100°C or 10 min at 85°C. you can use this as the template for PCR (as direct or by diluting). It works well for me. It doesn't affect my sequencing results.
Yes. I have tried the method and its very efficient and time saving
Also you can made a PCR direct from the bacteria colony, it is usefyl for amplification and sequencing
I am using heat lysis for DNA extraction by boiling few colonies in sterile water at 90 C for 20 min , followed b centrifugation at 13k rpm for 5min. finally collecting the supernatant.
but when i try to visualise the DNA on Gel , no DNA is seen. please suggest
Try this,
Re-suspended two colonies of bacteria in 200 µL, incubate at −80°C to freeze for 10 min and then boiled in water bath at 100 °C for 5 min. Repeat the procedure thrice then cooling on ice for 5 min. Centrifuge at 16,000 × g for 5 min, then pippete the supernatant into the clean tube.
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