Hello, I have some experience extracting plasmid DNA from Gram positives with the following protocol:

-Incubate the cells for 20 min at 55 C in 10 mM Tris pH 8.0, 50 mM NaCl, 10 mM EDTA, 20% sucrose and 10 mg/ml lysozym.

-continue according to the protocol of a regular plasmid extraction kit.

Hope this info will help you

Good luck!

Hello, I have some experience extracting plasmid DNA from Gram positives with the following protocol:

-Incubate the cells for 20 min at 55 C in 10 mM Tris pH 8.0, 50 mM NaCl, 10 mM EDTA, 20% sucrose and 10 mg/ml lysozym.

-continue according to the protocol of a regular plasmid extraction kit.

Hope this info will help you

Good luck!

Hello

Thank you for your feedback.

I have done something similar to what you suggested with slight variations.

I incubated cells with 20 ul of 50mg/ml lysozyme (prepared in TE buffer) for 30 mins at 37 degree C .

I suspect either the concentration of lysozyme or the incubataion temp are not sufficient to induce complete lysis

1. 50mg/ml the concentration of lysozyme in final lysis solution or

2. you add 20ul of 50mg/ml lysozyme solution on lysys solution.

If second please provide info about total volume of lysis solution.

I use vivantis plasmid extraction kit

According to the manufacturer's instructions, I added 250 ul of S1 solution to the pellets. I carried out additional step which was adding 20 ul of the 50 mg/ml lysozyme into the pellets resuspended in S1.

So the total volume became 270 ul. So the final conc of lysozyme becomes around 3.7 mg/ml.

I have come across few journals reporting plasmid extraction from bacillus ( other than b. cereus). In their studies they used lysozyme at final conc of 1 mg/ml and incubation time at 37 degree C for as short as 10 mins upto as long as 90 mins.

Yes, the most of publications concentration of lysozyme is 1mg/ml in final lysis solution. But also concentration depends from quantity of cells on which enzyme acting. So 1mg/ml is for about 10mg of biomass of bacilli.

So, if the final conc of lysozyme I used is higher, it means the lysis process should occur more efficiently right?

Most of the journals also suggested incubation with lysozyme at 37 degree C.

You suggested 55 degree. May I know the reason?

And another question is, would prolonging incubation time upto 1 hour be a good idea?

Hello, if you decreasing temperature you should increase incubation time.

You can use 20min at 50*C or 1 hour at 37*C as you wish.

55oC is giving you maximum speed but be careful above 55*C you will start to lose enzyme activity due to partially degradation and denaturation of enzyme.