You can also use mutanolysan, as an enzyme to lyse cells. I usually will take around 1 ml of cells, and resuspend them in a buffer containing 20mM Tris-HCl, 2mM EDTA, 1.2% Triton X-100, and then I add 1500 u/ml of mutanolysin, and 20 mg/ml lysozyme. Incubate at 37C for 1-2 hours. Another normal technique for lysing G+ cells is a freezing the cells then thawing them. Hopefully these methods will help.

Use this protocol

http://my.jgi.doe.gov/general/protocols/JGI-Bacterial-DNA-isolation-CTAB-Protocol-2012.pdf

You can add lisostaphin if your bug is a Staph. You could also just lyse bacteria and then proceed on the columns, but you have to check that your bacteria are really lysed and try not to overload the column

I would try to use CTAB like in plant genomic DNA extraction procedures. Usually people use if for plant tissues but it did work good for me also for gDNA extraction from drosophila.

I use fast prep beads to lyse Streptococcus pyogenes. It is fast, thorough, and efficient. After that, I use qiagen DNA kit to purify genomic DNA. It never disappointed me.

Try this protocol, it gives me good results (high molecular weight/high yield):

Bacterial gDNA extraction

Before starting:

Turn the water bath on (50 oC)

Thaw RNase A and Proteinase K on ice for 10 minutes

Precool glass beads (small diameters), plastic pestle and extraction buffer on ice for 10 minutes

1- Collect bacterial cells from overnight culture by centrifugation / or transfer small amount from the bacterial culture grown on LB agar into a 1.5 ml sterile Eppendorf tube and add small “equal” amount of the glass beads (small size).

2- Add 600 µl extraction/lysis buffer (200 mM Tris-HCl; 25 mM EDTA; 250 Mm NaCl; 0.5 % CTAB) and grind with the plastic pestle for few minutes.

3- Then add the following reagents:

~ 500 µg/ml RNase A (3 µl from 100mn/ml stock) >>>>> RT for 5 min

2% final volume SDS (66 µl from 20% stock);

450 µg/ml Prot. K (30 µl from 10 mg/ml stock);

2 mg/ml Lysozyme (15 µl from 100 mg/ml stock);

4- Mix by inverting the tube several times and incubate at 50 oC for 30 minutes.

5- Add equal volume chloroform (~ 0.7 ml); mix by inverting tube several times and incubate at room temp. (RT) for 10 minutes.

6- Spin down at 8000 rpm/ RT for 20 minutes for phase separation.

7- Transfer the aqueous phase (~ 400-500 µl, contains DNA) to new tube and spin down at max speed to precipitate any debris.

8- Precipitate the DNA from the clear lysate as follows:

Add 2 volumes 95% EtOH to the sample

Add 0.1 volumes 3M Na-Acetate

Mix by inverting several times

Incubate on ice or -20 oC for 20 minutes

Spin down at max for 10 minutes

Wash with EtOH 70%

Discard supernatant and air dry the pellet at RT and resuspend in water.

Dissolve the DNA pellet overnight on a shaker or at 55 oC for 1-2 hours.

GOOD LUCK

Characterization of a novel vip3-type gene from Bacillus thuringiensis and evidence of its presence on a large plasmid. FEMS Microbiol. Lett. 244, 353-358

Use Promega Wizard DNA isolation and purification kit. We are using it to isolate DNA from Clostridium, and Geobacillus for Genome sequencing

You can use of lysis solution for longer time.

all above methods are good, but in my view, gram positive or negative DNA, you can obtained by taken a loopful of bacterial colony and mixed with 500ul distal water, place it in water bath at 100C for 30 minutes, after that transfer to high speed centrifugation at 12,0000 rpm for 2 m. finally 3 ul of (DNA) at the upper surface of the solution collected carefully and you can use that DNA for PCR reaction

I use the protocol used by HAIN Life Science, Suspend colonies in 1000ul sterile water/ molecular grade water, spin in micro centrifuge at 10000rpm 15min, decant supernatant and add 100ul molecular grade water, place in heating block at 95C for 15 min, place in ultra sonicator for 15 min, spin in micro centrifuge at 13000 5 min then use supernatant for DNA.

Here i give the protocol which can used. I am sure that will be solve your problem..

Mini prep solution used for DNA isolation:

Solution I (suspension buffer):

Tris (pH 7.4-7.6) 50 mM

EDTA 1 mM

RNAs 100 µl/ml

Solution II (Denaturation solution):

NaOH 0.2N

SDS 1%

Solution III (Neutralization solution):

Potassium acetate 3M

(Adjust pH to 4.8-5.0)

DNA isolation method:

Protocol was used for the isolation of DNA from E.coli:

1) A single colony of each strain of bacteria ( S. aureus) was cultured in 20 ml liquid LB medium and grown overnight at 37 C.

2) The E.coli culture was centrifuged in 1.5 ml eppendorf tube at 14000 rpm for 5 minutes in Eppendorf® centrifuge (Model 5415 C Germany) at room temperature.

3) The supernatant was discarded and the pellet was allowed to dry for 2 minutes.

4) 100-μl solution I (Mini prep solution) was added to eppendorf tube and the pellet was suspended in the solution with the help of vortex (Vortex-2 Genie Scientific Industries, USA).

5) 150-μl solution II (Appendix III) was added to eppendorf tube and mixed well by inverting gently.

6) 200-μl of solution III (Mini prep solution) was added to eppendorf tube, mixed well and centrifuged at 14000 rpm for 5 minutes in the same centrifuge.

7) The supernatant was taken in fresh eppendorf tube and two volume of 100% ethanol were added.

8) Eppendorf was kept at -40 C for 30 minutes and centrifuged at 14000 rpm for 10 minutes in the same centrifuge.

9) The supernatant was discarded and the pellet was washed with 70% ethanol.

10) After centrifugation supernatant was discarded and pellet was vacuum dried.

11) 20 μl of sterile distilled water was added to the pellet to dissolve DNA and was stored at -40 C. The DNA is ready to use for the PCR.

It really depends if you are doing quantitative or qualitative analysis. For qualitative Analysis most of the above mentioned methods will do fine. However for quantitative Analysis I would recommend to incubate your samples overnight with proteinase K. We always use the Machery-Nagel TissueKit (with the Gram+ support protocol) and normally get recoveries between 90-95%. If you have samples with unknown bacterial concentration I would also recommend the use of an extraction control.

Thank you very much for all replies. I don´t want heat samples to 95 or 100ºC because I use gDNA for Whole genome sequencing experiments. I think mutanolysin o CTAB are de best options. I Incubated at 37C for 1-2 hours and them freezing cells with E.coli for plasmid isolation. But in this case I want isolation streptococcus gDNA.

You may like to check this paper, it will help you.

"Real time PCR of Nor~1 (aflD) gene of aflatoxin producing fungi and its correlative quantization to aflatoxin levels in South African compound feeds"

H E Iheanacho, M F Dutton, P A Steenkamp, L Steenkamp, H A Makun, A Swart, J Q Mthombeni. Journal of Microbiological Methods 97 (2014) 63–67

Please have a look over the attached article. Hope to be useful.

Good luck

Some gram-positive bacteria, particularly coagulase-negative staphylococci have very refractory cell walls. I recommend lysostaphin and a digestion time of at least one hour, but you might want to use a mixture of lysostaphin and lysozyme, plus any other cell wall degrading enzyme you can get hold of.

If, after throwing everything you have at them, the cell suspensions remain as non-viscous as before, take comfort at least in the knowledge that you are not the first to experience the frustration. I used to want to shrink myself and dive in and attack them with an axe!

The protocol I provided is actually for Streptococcus bacteria. We isolate genomic DNA from Streptococcal DNA and then use them for quantitative PCR. I hope the mutanolysin works for you, and if you have any more questions, feel free to ask. Good luck!

Lysozyme solution with Triton-X should do the trick. And I would recommend you try the gram+ protocol using the Qiagen QIAamp DNA Mini Kit. I use it with Mycobacterium and get good DNA considering how little sample I have to work with.

Yeastern biotech kit using lysozyme and triton x also can be used for gram positive bacteria.

You may also use proteinase k and ctab is the best conventional protocol.

By the way... do remember not to add the proteinase K before the other enzymes have done their work, as it will degrade them. I know it's obvious, but it's surprising how many people forget that proteinase K digests enzymes too.

Hi, I worked a lot with gDNA from Streptococcus and Enterococcus. Don`t use lysostaphin because is for Staphylococcus. It`s normal that the yield from Enterococcus is much more than Streptococcus. However for Streptococcus I had a discrete yield using the same protocol that is a phenol-chloroform protocol for high % GC. Key steps: Lysozime 0,5-1h 37 C, freeze-thaw 2 times, RNAseA, Proteinase K.

Thank you very much!!! I had good isolations results!!! 225ng/ul !!!

Good to hear that. Any idea what it was that helped?

Hi Alberto,

My name is Robert Deyes and I work with Promega's Technical Support group. you are certainly using the right preprocessing reagents. The lysostaphin cleaves the Peptidoglycan chains in the bacterial cell wall allowing for more efficient lysis. So I would certainly continue to use it as per the protocol that we indicate. I don't know whether you have mentioned it in your description of your work. However, one thing that is clearly important to maximize yields is to achieve an appropriate density of live, dividing cells (10exp8-10exp9 cells per ml although this clearly varies with bacterial strain). You may determine cell density by measuring the absorbance of the culture at 600 nm and comparing to a standard.

Total yields are expected to be in the 5-15ug of genomic DNA for that size of culture.

Many thanks and I look forward to hearing from you shortly Alberto.

Robert

The better and easy method for DNA extraction is benzyl chloride method. It's good working for GRAM+ bacteria. 

 Hi I am facing similar problems with genomic DNA extraction from s.pyogenes. May i know if you could share the optimised protocol that works for you with me?

I want to know it is necessary to use Lysostaphin for RNA extraction from enterococci ?

Thanks Dr. Stephen Anthony Jackson

Could you share the method for me? ;'-( I have a same problem with you.