Northern witth oligos

A. John Watson watson_j at bms.com
Sat Feb 24 12:04:52 EST 1996


In article <aquilla.1175496623H at emba-news.emba.uvm.edu>,
aquilla at salus.med.uvm.edu (Tracy Aquilla) wrote:

> In Article <4gf7l0$42m at newsbf02.news.aol.com>, sehuang at aol.com (SEHuang)
wrote:
> >Has anybody tried to hybridize northern blots with oligos (eg T4 kinase
> >labelled)
> >instead of using cDNA (random primed-labelled) ??
> >
> >How is specificity, sensitivity ??
> >
> >thanks for any comment,
> >
> >S.E.H.
> 
> I've done it before and it does work. However, I suggest making the oligos
> as long as you can, preferably about 40 nt. Also, it's very easy to
> over-wash and quickly remove all the signal. I wash the blots at room temp
> and vary the salt concentration to adjust stringency. If the oligos aren't
> long enough or specific enough, the background can be high, but if you keep
> washing to reduce the background, you'll lose the signal.
> Cross-hybridization to rRNA bands can also be a problem if you aren't
> careful about oligo design. Good luck.
>     Tracy

I have done also, and concur with Tracy's suggestions --  mostly.  I
prehyb and hyb in 5X SSPE/5X Den's/0.5% SDS at 42 C and wash 3x 5 min at
RT in 6X SSC/0.1% SDS and once for 1 min at the Tm (I calculate as [69.3 +
0.41(%GC)] - 650/L) in the same buffer.   Sensitivity is of course lower
than for a random-primed probe to the same transcript.

Cheers,

AJW

----------------
John Watson
Bristol-Myers Squibb Co.
watson_j at bms.com
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