DNA extration from bacteria using kits or conventional methods - (Jun/23/2012 )
Dear all, please i need ur recomendations for isolating DNA from bacterial culture (plates) both gram -&+ . I am using promega DNA purification kit and i followed all the steps and trouble shooting , but still i got very low concentration of DNA , i dont know why?? Do u recommend any other kits that were succssful with your bacteria?? Or do u have any conventional method to do?? Please i need detaild procedure coz an still new in this field
what are your downstream works? I used to suspend Streps from blood agar and simply boil it for 5 minutes, spin it down and take the supernatant. I get good concentration and purity as determined by the nanodrop (60ng/uL, 260/280 around 1.8). The genomic DNA is fine for all the PCR and sequencing I do.....
Let me know if you need more specific protocols if you don't mind it "not using a kit"
my downstream work will be identification by 16s sequensing. my DNA concentration is very low with poor quality, cutulres were optained originally from fish samples and they got the pure cutlres and identified them using API but i need to confirm them using sequencing, and am running out of time.
please if you don't mind, provide me with more specific detailed protocols (not kits) to allow the DNA extraction from both G+and G- .
youe help will be highly appreciated
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if you just need to do 16S sequencing, it is often suficcient to simply add a small amount of bacteria - we usually grow them in liquid media (500µL in Eppendorf tubes) for a few hours depending on the growth rate of the strain we are interested in and then add 1µL of this culture to the PCR mix without any further purification, but use 5 min (to 10 min depending on the Enzyme used) initial denaturation at 98°C combined with a hot start, high fidelity DNA Polymerase.
In 95% of the cases the PCR product is suitable for sequencing and giving good results.
But when doing this you must be absolutely sure that you have a pure culture - becuase otherwise you might pick up a contamination which is working better with your primers or is easier disrupted druing the temperature step!
Agree with geb.
Alternatively, you can just suspend your bacteria colony from plate into sterile water or TE buffer, boil for 5 minutes, spin down the debris and use the clear supernatant as your template for PCR.
16S PCR is not difficult, no worries.
p/s: we encourage everyone to share their protocol on the bulletin.