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Re: RNA isolation problems
Posted by: priyanka singh (IP Hidden, Unregistered user, )
Date: May 31, 2005 05:31AM
i have precipitated RNA initially at -20 degrees,but now when i am doing so for 10 mins at RT,as recommended, i am getting much better results..and even at -20, 30 min incubation is fair enough..
Re: RNA isolation problems
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: May 31, 2005 12:58PM
good! glad to hear it's going better!
Re: RNA isolation problems
Posted by: Valia (IP Hidden, Unregistered user, )
Date: June 1, 2005 12:11PM
I'm trying to cloning miRNA from mouse. I need to make the gel purification of miRNA. I have tested many differents methods, for example: difussion with buffer containing EDTA, SDS, etc., dialisys, kits to gel purify for DNA, but none of them have worked in my case. Please, could you suggest me other methods more efficient?.
Re: RNA isolation problems
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: June 1, 2005 02:05PM
Excise the RNA band of interest directly from your gel. Be sure to include a little extra gel above and below this band (~3mm if possible). Homogenize the gel pieces in a microcentrifuge tube by grinding them with a small glass rod or a 1000ul tip melted closed (briefly pass the tip over a bunsen burner and while the plastic is still soft, press the end closed by gently rolling the very tip on a flat surface). Be certain to grind the gel up into very small fragments. Add 4-5 times the volume of the gel slice of 20mM Tris, 1mM EDTA, 0.4M Acetate and 0.5% SDS to the tube and agitate it vigorously for at least 2 hrs at room temperature. You may get better extraction if you incubate it overnight. Spin the gel slice homogenate through a filter capable of trapping acrylamide gel fragments. Add 1ul of RNase free glycogen (Invitrogen Cat. #10814) and 3 volumes of 100% Ethanol. Cool to -20C for 25 minutes, centrifuge at 4C for 10 minutes to pellet the RNA (centrifuge speeds may vary to obtain this pellet). Decant the supernatant and wash the pellet in 70% ethanol. Allow the pellet to dry almost completely, then resuspend in water and store at -70C. You may wish to repeat the wash step 3x.
RNA isolation problems
Posted by: priyanka singh (IP Hidden, Unregistered user, )
Date: June 22, 2005 08:32AM
i am extracting RNA from drosophila heads..i have read people get as much as 1microgram RNA from single fly head...whereas i am getting from about 100 heads ,only 5-40 microgram yields.can anybody tell me where can i be loosing the RNA during extraction.i am doing exactly as per the trizol protocol.also what is the optimum amount of RNA to be used for one microarray hybridization.currently i m taking 10 micrograms for one slide.
Re: RNA isolation problems
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: June 22, 2005 04:00PM
If you haven't already tried it, use the Trizol modification for extracting RNA from small quantities of tissue (1 to mg). To do so: Add 800ul of Trizol to the tissue. Homogenize the sample as usual. *To reduce viscosity, sheer the genomic DNA with 2-3 passes through a 26 gauge needle prior to chloroform addition.* Add chloroform and proceed with the phase separation described in step 2 of your Trizol protocol. Prior to precipitating the RNA with isopropyl alcohol, add 5-10ug RNase-free glycogen (Cat. No. 10814) as a carrier to the aqueous phase. To reduce viscosity, sheer the genomic DNA with 2-3 passes through a 26 gauge needle prior to chloroform addition. The glycogen remains in the aqueous phase and is co-precipitated with the RNA. It does not inhibit first-strand synthesis at concentrations up to 4mg/ml and does not inhibit PCR. *Optional
Re: RNA isolation problems
Posted by: satinder singh (IP Hidden, Unregistered user, )
Date: July 4, 2005 03:34AM
i am extracting RNA from liver samples of swiss albino mice using tri reagent. after addition of isopropanol i am getting milky white curd like precipitates which subsequently forms a thick chalky white pellet. that pellet after washing with 75%ethanol is hard to dissolve in RNAse free water. also i am very carefully avoiding interphase, still A260/A280 ratio i am getting is very low. can anybody help me in this regard.
Re: RNA isolation problems
Posted by: oghalaie (IP Hidden, Unregistered user, )
Date: July 26, 2005 12:32AM
Hi
I am working on cytokine expression, and extracted RNA from tissue and I have some nonspesific band in PCR How I can dissolve this problem please ?
Re: RNA isolation problems
Posted by: kumarsysu (IP Hidden, New member, 9)
Date: August 14, 2005 09:54PM
You may loose RNA during maceration or during final step.
you can standarise by varying all the time and concentration at every step. bye and all the best
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