getting rid of RNA

[muntedkowhai]

So I'm bulking up my DNA constructs and I ran them on the gel today.

I saw RNA in one of my lanes.

my co worker told me i could use phenol and some washes to get rid of RNA or use a kit she had gotten from a biotech company.

i chose the latter because, hey its phenol.

i went thru all the steps but after a few washes, i did not see any pellet and when i put it thru the nanodrop. it came out with negative nanograms per microliter.

ok not good.

so where the hell in the washing steps could have gone wrong?

i was careful in the washing, i pippett only the SN and nothing more.

the only thing i can think of is, i left the solution in over 2 minutes the suggested time.

the solution was the kits' ligase wash.

so i dontknow if that would have done it.

i mean before i did this, i had 1050 ng/ul of dna and rna in the sample.

any suggestions?

[badchad]

Interesting problem. I'm no expert, but I've isolated RNA before. Generally speaking, RNA is very unstable. Usually, people trying to isolate RNA have the opposite problem (e.g. it's constantly degrading, and you have to use all sorts of RNAse inhibitors and such). I would think you could easily degrade the RNA simply by touching it. I guess this wasn't much help....

[blike]

i left the solution in over 2 minutes the suggested time.

That's probably not it. If it was that time sensitive there would be warnings somewhere.

 

Is there a particular reason you didn't go with RNAse/phenol wash?

[muntedkowhai]

well this was a kit you see, its less cumbersome then working with phenol, as my co worker said.

but alas, i lost the dna constructs in that eppie, so theres really nothing i can do now.

:<

[ed84c]

Isnt this the one with a Kiwi fruit and some washing up liquid? Maybe try that ?

[muntedkowhai]

thanks guys,

i think i'll just re precipitate the dna construct, if theres any of it left.

then run the gel on it again.

well i'll just precipitate it first and then see if theres even anything there.