Hi all, I am trying to isolate DNA from Mtb and I have been following two protocols:
1 ml liquid culture, spin and discard supernatent, dissolve pellet in 1X TE, heat at 80 degree Celsius for 15 mins, bring down temperature to 37 degree Celsius add lysozyme and keep in 37 degree celcius for 30 mins, after that I am following two steps
1) add AL buffer and prot K keep in 56 degree for 2 hr and follow other steps of Quiagen DNeasy kit
2) add 500 micro Lysis Lysis buffer plus prot K plus SDS keep in 56 degree for two hour. Add PCI, CI and then ethanol precipitation.
Following both the steps the recoveey has been low. Please suggest how shall I modify to get good yield. Anticipating some quick good suggestion from experts.
You are using Mtb H37Ra or Mtb Rv culture? Usually this bacterium grows well as a surface pellicle culture when you float is at the surface of sterile broth Middlebrook or Youmans & Youmans. What matters is the initial cell mass/ pellet that you have taken for lysis to release DNA.
If the specific equipment available, try bead beating for cell lysis step.
Regards
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