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RNA Isolation


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#1 weimin42

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Posted 14 April 2004 - 06:06 PM

Anyone in there are doing microarray research on B.japonicum USDA 110, i induced this bacterial strain with soybean seed extract (SSE) for 12 hours. When collecting the bacteria for RNA isolation, it was surprised that the induced bacterias differed with controled bacterias, like white cotton or fatty tissue, and uneasy to spin down even at 15000 rpm for 30 min. the most of bad thing is that the total RNA could not be successfully isolated.
I dont know what happened with SSE induction. anyone would like give some suggestions about it.

#2 phdconsult

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Posted 14 April 2004 - 07:04 PM

The fibrous matter you get with SSE induction are B. Japonicum surrounded with excess bacterial lipid- happens sometimes with SSE induction for unknown reasons. You lost the RNA when excess time was spent attempting to spin the cells down. Do not centrifuge, it is not going to work, trap the cells on filter- good luck

#3 weimin42

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Posted 14 April 2004 - 09:01 PM

Hi, Thanks for your reply.

In order to get RNA as much as possible, the bacterias were grown in the 200ml of YMB medium, so i must collect it by centrifugation. I will try to do it with higher rpm in a shorter time period.

#4 phdconsult

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Posted 15 April 2004 - 05:05 AM

You can try it - it may not work. Centrifugation, even ultracentrifugation does not help much to settle bacterial cells with lipid coating. Plan on a Whatman-3 filtration under gravity, or using a mild nonionic detergent for centrifugation or running a glycerol gradient where you can skim the cells off the top. Good luck and God Bless

#5 weimin42

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Posted 15 April 2004 - 05:02 PM

Hi,phdconsult.

I isolated the RNA yesterday and found that unbelievable yields of pellets came out after precipitation with isopropanol ,but it was not completely resuspended in RNase-free water.
The gel-running clearly showed the band of 23S and 16S, but the band before 16S made me disppointed . I was not sure if the mRNA had already degraded , so i want to ask how to know it ?

#6 phdconsult

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Posted 15 April 2004 - 07:05 PM

Weimin

Excellent!

Go ahead and proceed with your experiment. Don't worry about what you see (or don't see) on the gel. It is a bacterial population, your cells are in mixed growth phase (some are in early log phase, some are in late stat phase, some are in death phase), the nett mRNA content per cell is going to be highly variable, so yields are going to be low. However your photo or radio probes will work easily enough- start with a dot blot.

Good luck and God Bless




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