RNA Probe precipitation - (Apr/22/2010 )
I am trying to make an RNA DIG labelled probe at the minute. I am following the protocol to the letter but my yield is extremely low. I think its the precipitation that isnt working-I am using LiCl and ethanol but Im not seeing the white pellet that is supposed to be obvious.........
can anyone help...please
I also have no control DNA (that came with the kit) left, could I use the other control DNA even though its not the same plasmid???
What vector is you probe sequence cloned into? You should use that vector as your control (make some glycerol stocks, so when you run out of prepped DNA you can easily grow some more).
You can use a carrier to precipitate more efficently - denatured salmon sperm DNA, tRNA, or linear poly-acrylamide should do the trick.
The sequence is cloned in sPT19 plasmid, which I have more of but I was using the control DNA that came with the kit, could I grow up some of this in ecoli?? I was wondering if I could use RNA spin columns to clean up the RNA probe
I see, the control DNA was a positive control for the reaction working? I don't usually worry about this control - it just uses up the DIG labelled dUTP faster. You can do a reaction control by having an un-labelled reaction, which should still give you a band on a gel. The band will migrate faster than a DIG labelled one, as the DIG retards migration due to its size.
Brilliant, thanks a million bob1
I just use the Qiagen RNeasy minelute kits to clean up my probes. There's a chapter in there that's specific to cleaning up labeled probes.
I have tried the RNA columns with the kit and it didnt work for me, any chance you could send me your protocol, I followed the one in the book but I cant find anything specific to probes in my handbook. Its driving me crazy at the minute because I have a very high concentration of probe before I precipitate it but just cant get it to pellet out, I have been keeping the supernatants and measuring them-my RNA probe still seems to be in solution as the concentration of the first supernatant is very large. Also kept the supernatants when I tried the column and the same thing happened-first supernatant was very large-end yield extremely low.
Any help would be greatly appreciated because Im going slowing insane-I want to move on and do my in-situ hybridization
The protocol is for "RNA cleanup" - It's on p. 56 in the RNeasy mini handbook 2006 version. I did a digestion with DNAseI for 30 mins at 37oC before I started doing the clean up this way. It worked all right for me.
Actually I was wondering if you could also help me. I'm pretty new to this and I'm getting signals in my sense probe samples (negative control) and I have no idea how to get rid of this signal. If you have any ideas I'd be super grateful.
Your are like my new favourite person because your just one step ahead of me, any chance your in Galway?
I chatted my supervisor about your problem and he told me what you could do. Firstly, are you getting staining with your antisense-if so are they comparable as in staining the same things. In this case you may need to check that its not the antibody itself-so essentially another negative control-antibody with no probe.
However, if your antisense for e.g is staining epithelium and blood vessels and your sense is staining just epithelium its unspecific staining. In this case you can increase the strigency of your hybridization-this is done with the SCC washes, essentially your sense shouldnt bind as well and by washing more strigent you remove it but leave the antisense. You can also decrease the temperature of hybridization-even 1 degree can make a difference-this makes it more difficult for the probe to bind-so your making it more difficult for the sense probe to bind (this can be done in the opposite way if you have poor antisense binding). I think you can also decrease the hybridization time, by right your sense shouldnt bind as quickly.
I think unfortunately this technique takes a good bit of playing with the protocol to get the right conditions.
Hi PCR Novice,
Thanks for asking your supervisor for me. ^^; I'll try all those things. I've decreased the concentration of the probes used at the moment and I already did an antibody check and there were no signals. I'm developing the colour today so hopefully I'll see what's going on. I'm hoping I wouldn't have to redesign my probes. At the moment, the antisense and the sense strands are staining at around the same areas so it I'm kinda scared of what the outcome will be.
Hehe... I'm in New Zealand. So no, not in Galway. ^^