Link to this post | posted 14 Mar, 2016 15:05
dwestholm	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. Thanks! Dan Westholm

Link to this post | posted 15 Mar, 2016 18:34

dedrick

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 Bekah Dedrick

Link to this post | posted 15 Mar, 2016 19:41
dwestholm	Thank you Bekah! Did you use standard volumes as outlined in the Qiagen kit?

Link to this post | posted 16 Mar, 2016 11:39
dedrick	Yes I did. I attached the protocol here. Bekah Dedrick 116Kb

Link to this post | posted 16 Mar, 2016 12:55
dwestholm	Thanks again Bekah. One more quick question. Did you use the MP FastPrep 24 Homogenizer with the lysing tubes, or did you just vortex?

Link to this post | posted 16 Mar, 2016 13:01
dedrick	We use a Mini-bead beater from Biospec Products, Inc. I'm not so sure vortexing would do the trick. Bekah Dedrick

Link to this post | posted 16 Mar, 2016 13:55
dwestholm	Thanks for your quick reply!