Help: Ribonuclease protection assays.

David L. Haviland, Ph.D. haviland at KIDS.WUSTL.EDU
Tue Apr 2 02:13:14 EST 1996


On Mon, 25 Mar 1996, Married with Children wrote:

> In recent months the RPA system (Ambion), that I have previously 
> successfully used, has taken to generating empty lanes.  This problem is 
> appears irrespective of target mRNA, riboprobe batch, RNA sample, 
> diposable plasticware batch, glassware and pipette cleanliness, etc.  
> Even new, unopened kits give the same result...sometimes it works, 
> sometimes not, generally for an entire set of assays.  Is there 
> some small but crucial detail that I'm overlooking?  ANY feedback would 
> be greatfully appreciated.

Personally, I have found it imperative that one compute the Ti to for 
48-52'C for the *expected* hybridizing probe and then correspondingly 
readjust the % formamide in order to hybridize in this range.  The 
formula for this can be found in maniatis.

The formula is fairly common and is found in Davis as well as Maniatis
(11.46) but needs two factors added for the Maniatis version.  Assuming you
found the formula, Pm is the percent mis-matches and is a negative number. 
Also the percent of formamide is multiplied by 0.65 and is also subtracted.
B is 675 and L is the length of your probe.  M is the 
concentration of NaCl (Molar).

So it would read Tm=16.6Log[M] + .41[Pgc] + 81.5- Pm -B/L -0.65[Pf]

I find too often that "kits" just say "do it" at 42'C without 
consideration of probe length and percent formamide.  Note that per this 
formula, the higher % formamide, the lower the Tm...  thus with a short 
probe of less that 150bp and 80% formamide, you might need a Tm down in 
the mid-30's which is basically useless.

Punching out the nubmers for my specific RNAse protection and readjusting 
temp and formamide conditions was the only way I succeeded in getting 
RNAse protections to work consistiently.

Hope this helps,
David

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