Dear colleagues, prompt please, which one lysis buffer you are using to isolate bacterial DNA from the sputum samples? The isolated DNA in my experiment (500 mM NaCl, 50 mM TrisHCl pH=8, 50 mM EDTA, 4% SDS) is not enough purified of mucus polysaccharides.
We used this lysis buffer with the bead mill for 3 minutes. (Please do not advise commercial kits for me, I'm interested in buffer recipe).
Our sample preparation to liquefy the sputum: we used 1 volume of sputazol, after the vortex mixer samples were incubated on a shaker for 15 minutes on 100 rpm (RT).
Next we centrifuged it at 13000g of 15 min. Then we added to the pellet 1 ml of mutanolysin 100 U / ml + 5 μl of lysozyme and put the sample on a shaker for 15 minutes at 100 rpm, incubated for 30 minutes at 37° C, then centrifuged it at 13000g for 15 minutes.
Thank you in advance for your help.
Yulia,
follow discussion at https://www.researchgate.net/post/What_is_the_best_way_to_isolate_bacterial_DNA_from_sputum_samples_cystic_fibrosis_for_detection_of_pathogen_S_aureus
Alex, thank you very much for your reply, I think it's great help to me in my work.
But as you think could I try to use CTAB buffer, because in this method the polysaccharides doesn't precipitate?
Yulia,
the standard "theoretical" method for DNA extraction include both SDS and CTAB with high salt because SDS binds lipids, LPS, proteins (charged +), and CTAB binds DNA (charged -). (the first step looks like: 50 mkl of sample, 400 mkl of TE Buffer, 30 mkl of 10% SDS, 100 mkl of CTAB 10%, 80 mkl of 3M NaCl (or different salt), 15 min at 65oC.
After removal SDS - binded proteins and lipids by phenol of chloroform you can precipitate polysaccharides by LiCl2 and extract DNA by EtOH.
But, magnetic or resin balls or membranes would make your task simple. I have isolated DNA from many high EPS samples using SDS-CTAB with good results, but some colleagues dislike this method prefering commercial kits.
Many thanks for the answer, at first I try to use the method with CTAB. I don't want to resort kits, because I have a very small number of samples. If I have to be isolated from a larger number of samples, I think I listened to your advice about the resin balls/ membrannes precipitation.
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