can one over-crosslink nucleic acids to a membrane

Paul N Hengen pnh at ncifcrf.gov
Tue Oct 7 10:40:01 EST 1997


Ed wrote:

> I know that people sometimes calibrate their UV source, which implies that
> either "too little" or "too much" cross-linking would be detrimental to a
> later hybridization. Let's just sake for the sake of argument (yeah...or
> let's say this scenario happened to a "friend" :)) that one should leave a
> blot crosslinking for way too long (umm....by about 40 minutes !!!!), are
> these blots toast?

I'd go ahead with it. I UV X-link for 10-20 min. using naked :-o DNA.
I've not had any problems. Your colonies are probably okay.

> it's just that I picked and patched about 300 colonies for this...

Aww, come on. 300 is not that bad. 10 plates x 30 colonies...nothin'!
With toothpicks, it can be done before lunch. Do it again and probe
both sets of colonies.

> and I REALLY would rather not have to re-do it unless I have to. But the
> thought of going through all that radioactivity jazz for the sake of blots
> that are not going hybridize is also a distasteful consideration...

Switch to chemiliuminescent detection for your colony blots. The probe
is much more stable, the washes can be tipped down the sink, and it
is not as distasteful (no robing up and/or double gloving/degloving).
It is VERY easy.

@article{Hengen1997Augtibs,
author = "P. N. Hengen",
title = "Methods and reagents - Chemiluminescent detection methods",
journal = "Trends in Biochemical Sciences",
volume = "22",
number = "8",
pages = "313-314",
month = "August",
year = "1997"}

-Paul.

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