Salting Out Genomic DNA Extraction Method
Isolation of high molecular weight (HMW) genomic DNA by salting out
The salting out method is used to isolate high yields of high molecular weight DNA, usually for applications such as whole genome sequencing. It is particularly useful for long read sequencing such as PacBIO and Nanopore, as Streptomyces has long regions of repetitive DNA sequences.
It is recommended that a whole day is allocated for this protocol, particularly if it is the first time conducting it.
Preparation and reagents needed
- Mycelium from 25ml culture (split a 50ml culture into 2)
- SET buffer:- 75mM NaCl, 25mM EDTA pH8, 20mM Tris-HCl pH7.5
- Lysozyme:- 50mg ml-1
- Proteinase K:- 20mg ml-1
- SDS:- 10%
- NaCl:- 5M
- 70% Ethanol
- TE buffer of sterilised dH2O
- Sealed Pateur pipette
- Eppendorf tubes
- 10ml pipettes
- Yellow tips/blue tips and P200/P1000 pipette
- 5ml or 10ml pipette
- Resuspend mycelium in 5ml SET buffer in a 50ml falcon tube. Add 100µl of lysozyme solution. Incubate at 37 degrees C° for 1-3 hours, lysis step is complete when the reaction has clarified and turned viscous. Note that the reaction may not clarify completely, this stage may require organism specific optimisation.
- Add 140µl proteinase K solution and 600µl 10% SDS, then mix by inversion. Incubate at 55° for 2 hours and invert occasionally
- Add 2ml 5M NaCl and mix by inversion
- Allow to temperature to cool to 37° or lower before adding 5ml chloroform. Mix by inversion for 30 minutes at 20°
- Centrifuge at 4500g for 15 minutes at 20°
- Carefully remove the top layer with a cut pipette tip and transfer to a clean tube. If the solution is still opaque, consider adding more NaCl, SDS and Chloroform as per steps 2-4 and repeat step 5
- The solution should be incubated on ice for 2 minutes, before adding 0.6 vol ice cold isopropanol. Mix by inversion and continue to incubate on ice for around 3 minutes. Spool DNA onto a sealed pasteur pipette
- Rinse DNA in 5ml 70% ethanol and leave to air dry. Using cut tips, resuspend in 500µl of TE or sterilised dH2O
Adapted from Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F. and Hopwood, D.A., 2000. Practical Streptomyces genetics (Vol. 291). Norwich: John Innes Foundation.
Protocol adapted for ActinoBase by Jake T. Newitt, PhD student at the University of East Anglia.