RNA extraction using TRIzol - (May/22/2009 )

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Yeah I tried the needle thing too. I saw the filamentous noodle floating around the bottom of the tube, so I don't know where it went. I even ran a gel and there was absolutely nothing. I think I might try using the aqueous layer from TRIzol on the minikits.

-Ahrenhase-

Hi,

I'm not too worry about the residual ethanol in RNA pellet. Air "drying" it for about 5 to 10 min is already sufficient. "Drying" here means allowing the 70% ethanol to evaporate to a conc of below 30-50% ethanol content. However, note that water will not evaporate as easily as ethanol.

Alternatively, heat briefly in a 50 degree C oven for say 1 to 2 min. Then air dry, allowing EtOH to evaporate while residual water remain in the tube. There'll be droplets at the side of tube and if you are really particular about completely eliminating all droplets, use a clean tissue to blot out these droplets.

However, I sincerely believe that your low A260/A230 is due to guanidinium carryover. I've had the same problem and after a second ethanol precipitation, the ratio is at a very healthy level. Example, before EtOH precipitation I'll get around 0.9 to 1.5 reading, now... I'm getting >2.0.

Good luck.

P.s. there's a link for troubleshooting low a260/a230 for rna @ http://wheat.pw.usda.gov/~lazo/methods/pro/tb087.html. NOTE: I'm using Trizol for RNA isolation.

-lsek-

Sounds like you're being TOO delicate...need to work fast and without concern with RNA but in an RNAse-free zone.

Having done 1000's of RNA preps using TRIzol or RNeasy etc. I recommend not using TRIzol. RNeasy with on-column DNAse digestion is far superior and doesn't require nasty extractions with phenol. Total RNA preps using RNeasy consistently outperforms TRIzol if your RNA is for qRT-PCR or microarray, but equal in northerns.

-eldon-

eldon on May 28 2009, 07:28 PM said:

But don't you lose all the miRNAs with the RNeasy columns?

Clare

-Clare-

But don't you lose all the miRNAs with the RNeasy columns?

Clare

No.

-eldon-

unfortunately you do lose the small RNA's with most column-based RNA isolations (including the RNeasy columns)!

-ien-

phage434 on May 26 2009, 01:48 PM said:

TE is definitely NOT out of the question. The only problem with TE is the (very small) amount of EDTA, 1 mM. When you set up your PCR reaction, this is diluted at least 20x, so you end up with 50 uM EDTA in your PCR reaction, which will hardly affect it at all, given the 1-2 mM magnesium in a typical buffer.

The EDTA will not inactivate RNAses, so it will not have the benefits that it has in DNA storage, where DNAses are inactivated by chelation of Mg++.

If you insist on eliminating the EDTA, then use 10 mM Tris pH 7.5 to dissolve your RNA. Water is a poor second choice.

I agree. A TE buffer with 0.1 mM EDTA is also a good option.

-ien-

can somebody help me...i have a difficulty in extracting RNA from bull sperms. i haver tried it before and no RNA bands visualized during gel. the OD reading also very low..any Method regarding this matter? please help..huhuhu...

-faiz-

have anybody have experience working with bull sperm in extracting the RNa USING tRIZOL?

-faiz-

faiz on Jan 26 2010, 12:02 AM said:

have anybody have experience working with bull sperm in extracting the RNa USING tRIZOL?

You might want to make a new topic as this is quite an old post

Clare

-Clare-

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