Hi Jacob,

I am using the AllPrep kit to extract DNA and RNA from a mixed bacterial community. My cell lysis protocol uses a lysozyme treatment (200 ul) followed by bead beating in RLT plus (700 ul) using stainless steel beads. The tech rep I talked to at Qiagen strongly discouraged me from using glass beads, as the RLT plus buffer modifies the DNA so that it adheres to silica (which is what the DNA column is made of). I've had good luck getting DNA and RNA, but the DNA is consistently "dirty" and I need to take it through a ethanol clean-up to get decent 260/280 ratios.

I'd be happy to send you more specific information if you are interested.

Best,

Mary          

I haven't used this product, but while using TRI reagent for cyanobacteria, i noticed that lysis was not efficient. So, i used to freeze-thaw the samples with the TRI reagent and then use 0.5 mm glass beads and vortex which improved my extractions drastically. You may refer to the following article for description of the method, i hope this is helpful: http://journal.frontiersin.org/Journal/10.3389/fmicb.2013.00374/full

Thank you for your response Sandeep. Bead beating should be an effective way of performing lysis in this case and I'll make sure to review your paper for your methods. Did you have any concerns with shearing DNA/RNA when you were doing this work?

Thanks again,

Jake

We used the RNA for Microarray and got pretty good results with that and got good RIN values with Bioanalyzer, so i am sure at least the RNA was not sheared. I actually did not extract DNA, though the TRI reagent allows you to do that from the same sample and also protein, hence the name TRI, i guess. In general, bead beating is considered pretty gentle as opposed to something like sonication. But we did not have a bead beater, so i just vortexed samples three times for 30 secs each with intermittent resting on ice for 30 secs.

Hi Jacob,

I do an incubated enzymatic lysis on a mixed bacterial sample ( G +, and G -), i do this after incubation in RNA protect batcerial reagent, it works pretty well, I would go along with Sandeep in recommending a repeat bead beating approach with ice cooling intermittenly, for the mechanical lysis, so as not to over heat the RNA strands. then ice, centrifuge, ice again and add supernatent to RLT buffer (with 1% beta-mercaptoethanol, note I used allpreps dna/rna mini, it cleaves di sulphide bonds so might be an issue if working with proteins), proceeding as normal for the rest of the protocol. It will prob require a lot of optimization for micro algae. Hope this was of some use to you.

Regards,

Conor

Hi Jacob,

I am using the AllPrep kit to extract DNA and RNA from a mixed bacterial community. My cell lysis protocol uses a lysozyme treatment (200 ul) followed by bead beating in RLT plus (700 ul) using stainless steel beads. The tech rep I talked to at Qiagen strongly discouraged me from using glass beads, as the RLT plus buffer modifies the DNA so that it adheres to silica (which is what the DNA column is made of). I've had good luck getting DNA and RNA, but the DNA is consistently "dirty" and I need to take it through a ethanol clean-up to get decent 260/280 ratios.

I'd be happy to send you more specific information if you are interested.

Best,

Mary          

Hello Mary J Hagen

I am trying to isolate DNA and RNA using All prep kit from prostate tissue biopsy samples, however, I am facing trouble in isolating RNA (although DNA is good).

Since, we do not have bead beating machine such as tissuelyser we use probe based homogenization method or syringe based homogenization in RLT plus buffer. Can you please help me troubleshoot this.

Thanks in advance.

Regards,

Vipul