Comments on: Homogenization and Bead Tube Methods for RNA Work http://www.mobio.com/blog/2010/06/18/homogenization-and-bead-tube-methods-for-rna-work/ Tue, 13 Dec 2011 00:32:47 +0000 http://wordpress.org/?v=2.9.1 hourly 1 By: Protect your RNA by Working Smarter and Faster « MO-BIO: The Culture Dish http://www.mobio.com/blog/2010/06/18/homogenization-and-bead-tube-methods-for-rna-work/comment-page-1/#comment-11133 Protect your RNA by Working Smarter and Faster « MO-BIO: The Culture Dish Fri, 30 Sep 2011 23:57:30 +0000 http://www.mobio.com/blog/?p=1044#comment-11133 [...] buffer (or phenol based lysis reagent) to your sample for homogenization.  We’ve discussed lysis and homogenization before, including a thorough overview of bead tubes and sample types.  Bead beating is a great way [...] [...] buffer (or phenol based lysis reagent) to your sample for homogenization.  We’ve discussed lysis and homogenization before, including a thorough overview of bead tubes and sample types.  Bead beating is a great way [...]

]]> By: Suzanne Kennedy http://www.mobio.com/blog/2010/06/18/homogenization-and-bead-tube-methods-for-rna-work/comment-page-1/#comment-5488 Suzanne Kennedy Sat, 14 Aug 2010 01:04:48 +0000 http://www.mobio.com/blog/?p=1044#comment-5488 Hi Pepe, Typically -80C storage will keep everything in stasis so there will be no RNase activity. Purified RNA can be kept at -80C for years. Plant tissue, however, may not be as stable. I would definitely give it a try. Adding beta mecraptoethanol to the lysis buffer will help kill RNases when you resuspend the powder. Proteinase K is a potent RNase inhibitor too. Best, Suzanne Hi Pepe,
Typically -80C storage will keep everything in stasis so there will be no RNase activity. Purified RNA can be kept at -80C for years. Plant tissue, however, may not be as stable.
I would definitely give it a try. Adding beta mecraptoethanol to the lysis buffer will help kill RNases when you resuspend the powder. Proteinase K is a potent RNase inhibitor too.

Best,
Suzanne

]]> By: pepe http://www.mobio.com/blog/2010/06/18/homogenization-and-bead-tube-methods-for-rna-work/comment-page-1/#comment-5486 pepe Sat, 14 Aug 2010 00:03:21 +0000 http://www.mobio.com/blog/?p=1044#comment-5486 how long can homogenized (pulverized/powdered) plant tissues be stored at -80 degrees celcius prior to RNA extraction? will the integrity of the RNA be preserved after prolonged storage at the said temperature? im asking because i ground some samples and stored them at -80 for a year. now i wanna extract RNA from these samples how long can homogenized (pulverized/powdered) plant tissues be stored at -80 degrees celcius prior to RNA extraction? will the integrity of the RNA be preserved after prolonged storage at the said temperature? im asking because i ground some samples and stored them at -80 for a year. now i wanna extract RNA from these samples

]]> By: Homogenization Tips: Choosing a Bead Tube « MO-BIO: The Culture Dish http://www.mobio.com/blog/2010/06/18/homogenization-and-bead-tube-methods-for-rna-work/comment-page-1/#comment-5479 Homogenization Tips: Choosing a Bead Tube « MO-BIO: The Culture Dish Fri, 13 Aug 2010 19:42:48 +0000 http://www.mobio.com/blog/?p=1044#comment-5479 [...] Homogenization and Bead Tube Methods for RNA Work [...] [...] Homogenization and Bead Tube Methods for RNA Work [...]

]]> By: rflp http://www.mobio.com/blog/2010/06/18/homogenization-and-bead-tube-methods-for-rna-work/comment-page-1/#comment-4813 rflp Tue, 03 Aug 2010 18:26:25 +0000 http://www.mobio.com/blog/?p=1044#comment-4813 your web site is great.I liked it, I bookmarked it and will examine detailed pages later, your web site is great.I liked it, I bookmarked it and will examine detailed pages later,

]]> By: Marcia http://www.mobio.com/blog/2010/06/18/homogenization-and-bead-tube-methods-for-rna-work/comment-page-1/#comment-3301 Marcia Thu, 01 Jul 2010 13:37:44 +0000 http://www.mobio.com/blog/?p=1044#comment-3301 Suzanne, I'm using ultra-high density beads (2.0 mm)and spin columns. All the materials that I use are RNase-free. I'm working with samples of Theobroma cacao (leaves) and, sometimes, it's difficult to pass the supernatant through column because the samples are very, very viscous. Thanks for your attention. Suzanne,

I’m using ultra-high density beads (2.0 mm)and spin columns. All the materials that I use are RNase-free. I’m working with samples of Theobroma cacao (leaves) and, sometimes, it’s difficult to pass the supernatant through column because the samples are very, very viscous.

Thanks for your attention.

]]> By: Suzanne Kennedy http://www.mobio.com/blog/2010/06/18/homogenization-and-bead-tube-methods-for-rna-work/comment-page-1/#comment-3287 Suzanne Kennedy Thu, 01 Jul 2010 02:24:17 +0000 http://www.mobio.com/blog/?p=1044#comment-3287 Hi Marcia, I think probably 10 minutes is too much after the same is already macerated, if you vortexed that long. What type of beads did you use for the vortex step- what size beads? You can try adding some beta-mercaptoethanol into the lysis buffer to protect from RNases. Use 10 ul per ml of lysis buffer. Have you tried grinding in liquid nitrogen, resuspend in lysis buffer, and then go right into the prep? If the sample is not viscous then it may be just fine. The genomic DNA causes a lot of viscosity that can make handling tough but maybe your sample is already broken down enough. If there is viscosity after adding lysis buffer, try running it through a 1 cc syringe with a 25 guage needle to break it down. Do you bake your mortar and pestle to make it RNase-free? Best, Suzanne Hi Marcia,
I think probably 10 minutes is too much after the same is already macerated, if you vortexed that long. What type of beads did you use for the vortex step- what size beads?

You can try adding some beta-mercaptoethanol into the lysis buffer to protect from RNases. Use 10 ul per ml of lysis buffer.
Have you tried grinding in liquid nitrogen, resuspend in lysis buffer, and then go right into the prep? If the sample is not viscous then it may be just fine. The genomic DNA causes a lot of viscosity that can make handling tough but maybe your sample is already broken down enough.
If there is viscosity after adding lysis buffer, try running it through a 1 cc syringe with a 25 guage needle to break it down.

Do you bake your mortar and pestle to make it RNase-free?

Best,
Suzanne

]]> By: Marcia http://www.mobio.com/blog/2010/06/18/homogenization-and-bead-tube-methods-for-rna-work/comment-page-1/#comment-3286 Marcia Thu, 01 Jul 2010 01:20:07 +0000 http://www.mobio.com/blog/?p=1044#comment-3286 Hi Suzanne, I'm working with plant tissue and I'm trying to extract RNA. In fact, I use the mortar and pestle with liquid nitrogen, I resuspend the sample in lysis buffer into the bead tube and shake with a vortex. But, when I did the electrophoresis looks like there is a RNA degradation. I macerated a lot until obtention of a very fine powder. Do you think this prolonged maceration followed by vortex could cause the RNA degradation, because the protocol that I used says that the sample must be finely minced instead of macerated. Thanks very much for the help. It's the first time I try to use the beads tube method. Hi Suzanne,

I’m working with plant tissue and I’m trying to extract RNA. In fact, I use the mortar and pestle with liquid nitrogen, I resuspend the sample in lysis buffer into the bead tube and shake with a vortex. But, when I did the electrophoresis looks like there is a RNA degradation. I macerated a lot until obtention of a very fine powder. Do you think this prolonged maceration followed by vortex could cause the RNA degradation, because the protocol that I used says that the sample must be finely minced instead of macerated.

Thanks very much for the help. It’s the first time I try to use the beads tube method.

]]> By: Suzanne Kennedy http://www.mobio.com/blog/2010/06/18/homogenization-and-bead-tube-methods-for-rna-work/comment-page-1/#comment-3218 Suzanne Kennedy Wed, 30 Jun 2010 03:57:55 +0000 http://www.mobio.com/blog/?p=1044#comment-3218 Hi Marcia, Thanks very much for your comment! When it comes to RNA, the options are limited. If you are working with bacteria or fungus, you can use the vortex for lysis. This is what we do in our lab. If you have animal or plant tissue, you have to first break the matrix of connective proteins holding the tissue together and then break the cells. So a lot of force is needed. What kind of samples are you working with? You could use the mortar and pestle with liquid nitrogen to first break down the tissue to a powder and then resuspend the powered sample in lysis buffer and add to a bead tube to finish the cell lysis and shear genomic DNA on a vortex. This should work. Let me know about the samples you work with and I can provide more advice. Best, Suzanne Hi Marcia,
Thanks very much for your comment! When it comes to RNA, the options are limited. If you are working with bacteria or fungus, you can use the vortex for lysis. This is what we do in our lab.
If you have animal or plant tissue, you have to first break the matrix of connective proteins holding the tissue together and then break the cells. So a lot of force is needed. What kind of samples are you working with?
You could use the mortar and pestle with liquid nitrogen to first break down the tissue to a powder and then resuspend the powered sample in lysis buffer and add to a bead tube to finish the cell lysis and shear genomic DNA on a vortex. This should work.

Let me know about the samples you work with and I can provide more advice.

Best,
Suzanne

]]> By: Marcia http://www.mobio.com/blog/2010/06/18/homogenization-and-bead-tube-methods-for-rna-work/comment-page-1/#comment-3209 Marcia Wed, 30 Jun 2010 02:53:44 +0000 http://www.mobio.com/blog/?p=1044#comment-3209 Hi Suzanne, First, I liked your post very much, because it solved a lot of doubts. Second, I'd like to known if I could use a vortex instead of the PowerLyzer, because I don't have in the laboratory a homogenizer for the beads tube. In this case, what would be the time required for the homogenization of the sample at the vortex? Thanks, Marcia Christina Hi Suzanne,

First, I liked your post very much, because it solved a lot of doubts.
Second, I’d like to known if I could use a vortex instead of the PowerLyzer, because I don’t have in the laboratory a homogenizer for the beads tube. In this case, what would be the time required for the homogenization of the sample at the vortex?

Thanks,

Marcia Christina

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