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RNA isolation protocol from smeg

| posted 14 Mar, 2016 15:05
Hello everyone,
Does anyone have a (cheap) RNA isolation protocol for smeg infections? I'd imagine the waxy cell wall would prove difficult for most standard commercially available kits.
Dan Westholm
| posted 15 Mar, 2016 18:34
Hi Dan,
Yep, we use the Qiagen RNeasy kit. It's super easy and relatively inexpensive.
The kit has you resuspend your cell pellet in a buffer called RLT. We place this suspension in a matrix lysing tube (matrix B - MP Biomedicals 6911-100) and bead beat for 45 seconds on max speed, two times (icing in between) to break open the cells. Then, you centrifuge this tube briefly and transfer the supernatent into a new tube and go on with the procedure where you add 560ul of 80% EtOH.
Let me know if you need more information!!
Bekah Dedrick
| posted 15 Mar, 2016 19:41
Thank you Bekah! Did you use standard volumes as outlined in the Qiagen kit?
| posted 16 Mar, 2016 11:39
Yes I did. I attached the protocol here.
Bekah Dedrick
| posted 16 Mar, 2016 12:55
Thanks again Bekah. One more quick question. Did you use the MP FastPrep 24 Homogenizer with the lysing tubes, or did you just vortex?
| posted 16 Mar, 2016 13:01
We use a Mini-bead beater from Biospec Products, Inc. I'm not so sure vortexing would do the trick.
Bekah Dedrick
| posted 16 Mar, 2016 13:55
Thanks for your quick reply!
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