I found some protocols on the internet, but I still confuse between lysis buffer for genomic and plasmid isolation. Thank you.
Hi Nguyen,
usually you start with an SDS buffer to lyse the bacteria and with proteinase K to get rid of the protein followed by some purification steps. Below is a short protocol but I am sure there are kits on the market so that you can avoid using phenol and chloroform. If you follow the protocol you may add an RNAse step.
- Grow bacterial strain to saturation.
- Spin bacteria in 1.5 ml for 2 min in microcentrifuge.
- Resuspend in 567ml TE buffer, 3 ml of 10% SDS, 3 ml of 20 mg/ml proteinase K. Mix and incubate 1 hr at 37°C.
- Add 100 ml of 5 M NaCl. Mix thoroughly.
- Add 80 ml of CTAB/NaCl solution (10% CTAB in 0.7 M NaCl). Mix. Incubate 10 min at 65°C.
- Extract with an equal volume of phenol/chloroform/isoamyl alcohol. Spin 5 min in microcentrifuge.
- Transfer aqueous phase to a fresh tube. Extract DNA with chloroform/isoamyl alcohol. Spin 5 min in microcentrifuge.
- Transfer aqueous phase to a fresh tube. Precipitate DNA with 0.6 vol isopropanol.
- Wash precipitate with 70% ethanol. Remove supernatant and briefly dry pellet.
- Resuspend pellet in 100 ml TE buffer.
Dear Nguyen,
Below is the link for the genomic DNA isolation kit.
https://www.thermofisher.com/order/catalog/product/K0512
Hope this helpful.
Uwe, please check you volumes. Sometimes I think "mL" should read "uL"!! If not, do you really use a total of 1.5 LITRES of phenol mix??
Plasmid DNA purification use supercoil structure of episoms. You do alkaline denaturation and selective removal of genomic DNA from plasmid - which cannot melt in alkaline solution. In current protocols of QIAgen - you can get plasmids practically without genomic But classical CsCl gradient with EtBr can used
DNA, If you want to separate plasmid and genomic DNA - Gel electrophoresis is a method you need
Hi Geoff,
Thanks! The "µ" was lost during copy/paste and then I tried to add it again. I was obviously careless. Liters of Phenol are no fun.
I hope I found all typos and the correction works now:
- Grow bacterial strain to saturation.
- Spin bacteria in 1.5 ml for 2 min in microcentrifuge.
- Resuspend in 567 µl TE buffer, 3 µl of 10% SDS, 3 µl of 20 mg/ml proteinase K. Mix and incubate 1 hr at 37°C.
- Add 100 µl of 5 M NaCl. Mix thoroughly.
- Add 80 µl of CTAB/NaCl solution (10% CTAB in 0.7 M NaCl). Mix. Incubate 10 min at 65°C.
- Extract with an equal volume of phenol/chloroform/isoamyl alcohol. Spin 5 min in microcentrifuge.
- Transfer aqueous phase to a fresh tube. Extract DNA with chloroform/isoamyl alcohol. Spin 5 min in microcentrifuge.
- Transfer aqueous phase to a fresh tube. Precipitate DNA with 0.6 vol isopropanol.
- Wash precipitate with 70% ethanol. Remove supernatant and briefly dry pellet.
- Resuspend pellet in 100 µl TE buffer.
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