Hi, I would advise to not do lysozyme digestion overnight, because all your extracted DNA is getting chewed up as they are being released from cells. Also, include your RNase at the same time; as you should try and get your DNA away from the cell debris sooner rather than later.

Possibly try using a CTAB protocol as well, as it will consistently give you good yields.

The main idea is to complete your total extraction within the same day.

53ng/L is very little DNA when you load microliters into agarose gel. That may be the issue. Other than that, if you have a faint band for positive control or dna ladder, that implies poor EtBr staining.  

If possible, let's switch to the bead extraction method, it is easy, fast and economic (finish a sample in 20 mins). This method has been working well for many members of Gram-positive bacteria, eg. Listeria, enterococcus, Leuconostoc, etc. The method can be found at :

http://www.ncbi.nlm.nih.gov/pubmed/15935495

or

http://www.sciencedirect.com/science/article/pii/S0167701205001077

Hi, I would advise to not do lysozyme digestion overnight, because all your extracted DNA is getting chewed up as they are being released from cells. Also, include your RNase at the same time; as you should try and get your DNA away from the cell debris sooner rather than later.

Possibly try using a CTAB protocol as well, as it will consistently give you good yields.

The main idea is to complete your total extraction within the same day.

Hi Francisco Lakay!... thanks for your answer, do you have any CTAB protocol that I can use? I have many and I do not know which will be the best for G-positive bacteria. 

Hi Graciana 

Just go through this link ...it may help you 

http://onlinelibrary.wiley.com/doi/10.1002/0471142727.mb0204s56/pdf

Genetic analysis of humans mainly relies on high yields of pure DNA samples.  The extracted DNA from the blood when subjected to PCR is often problematic, especially when protein and RNA contaminant remains during extraction. In order to overcome these problems  use Proteinase-K, during the DNA extraction method. Using this method, DNA was extracted from many samples using 5ml blood. The DNA purity ratio A260/A280 was 1.78-1.79 indicating good DNA levels supposed to be free from contamination. The technique is ideal for isolation of DNA from fresh blood. Thus the optimized protocol for DNA isolation is recommended for better results in PCR analysis, RFLP and further molecular analysis.

I agree with Mr Lakay. I tried including lysozyme for my buccal epithelial cells DNA extraction last time and the concentration is too low. It only improves after I remove it from my protocol.

If you need to stick to phenol:chloroform, perhaps you can try to homogenise the solution gently to prevent DNA shearing.

 Hi. I have attached the protocol we use to extract DNA from group A Streptococcus. It takes all day, but only one day, and has consistently yielded high quantities of good quality DNA (such that we typically perform subsequent PCR reactions using a 1:100 dilution of the prep). I hope it helps. Cheers.

Thanks Jessica!... I am trying it right now... but DNA precipitates did not apear. so I put it one hour at -20ºC.

Thanks again

Graciana

typically, during step #22 if there is no visible precipitate, I proceed with the protocol as if there was - eg, centrifuge and decant without changing tubes, presuming there is a tiny pellet that I can't quite visualize. Upon re-suspension, I have always gotten *some* DNA - and it's always been enough for our downstream applications. good luck.

Thanks!! have you meassured the amount of DNA? I need about 100 ng/ul.

Graciana...DNA is not always visible...Jessica is right..you can proceed and I am 99% sure that you will the desired concentration.Good luck.!