I have a spin column based kit, but I'm not sure on the initial step of obtaining and stabilising the bacilli colonies from LJ medium to continue with the kit protocol.
Or is it better to culture them in liquid media?
What do you mean with stabilising? If you could pick a bit of bacteria and culture it in liquid media it would be great to be sure you don't lose your sample. In the meanwhile (it is terrible how slow grows tuberculosis right?) you could pick bacteria from your LJ and then try to extract with your kit.
I would prepare in a screwed-cap tube 1ml of TE buffer with 0.05% of Tween and I would pick bacteria from LJ passing it to the tube. Once enough bacteria is loaded in the tube (but not too much since you could obstruct the columns) I would pipette (VERY CAREFULLY! ) up and down to homogenize the sample so you can disrupt the bacilli clumps.
Take care if your kit is not specific for tuberculosis, since this bacilli is very hard to lyse and it could give you very poor RNA yields.
Bests!
Yes kit method is better, Kit have column got high quality of RNA or you may try trizol method for trail your experiment
Good Luck!!!
Thank You Goig & Selvat. By stabilising, I meant keeping the RNA intact once I remove the colonies from the solid media, but I guess if I proceed immediately with the RNA extraction I wont need any RNA stabilising agent.
I'll try collecting the bacilli in TE and then proceed with the kit. If I dont get good quality RNA, then I will culture them again in liquid media.
And would Triton-X work in place of Tween?
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