[Histonet] Re: sticky column filter

From:Jimmy Lao

Hi, here let me take a chance saying thanks to Judith
and Debby Grant for their kindly giving me advice.
It seems to me the No.3) from Judith will help my
Even thought this histonet group traffic with huge
amount email, you getting something useful is most
Thanks again!
from a developmental Lab 
skirball institute
NYU medical center

--- Judith van Dommelen  wrote:
> Hello Jimmy,
> I saw your question on the internet. If you still
> have
> problems, try one of the following:
> 1) make a prerinse program with ethanol or methanol
> (whatever u use for rehydration) and rinse the
> columns
> before loading your embryos
> 2) Use heat permeation instead of proteinase K
> treatment: after rehydration, place embryos in fresh
> PBST in a 2 ml (cryo) tube, place them in a 95*C
> waterbath for 5 min. and then snapcool on ice.
> Replace
> again with fresh PBST and place embryos back in
> columns. I found that pK treatment damaged the
> embryos
> too much which made them more sticky
> 3) Cover the column filter with Sephadex: Dissolve a
> teaspoon of Sephadex G50 in 100 ml of deionized
> RNA-free water, solve properly and then fill columns
> till 1/3 of volume with the Sephadex solution. Let
> columns drain in the machine, a gooey layer of
> sephadex now covers the filter frit. This has no
> negative effect on signal development.
> Good Luck\
> Judith

We use 1% NaOH (Sodium Hydroxide) 6 hours to
overnight, then we rinse 
3x with distilled water, then soak them in distilled
H20 overnight, then 
rinse 2x with distilled H20, then dry them in an oven
@ 60 degrees 
Celsius, and rinse in 100% EtOH then dry again in the
oven @ 60 degrees 
Celsius. Usually we can use the columns until the
filters pop out from the 
columns, approximately 3-4 times. Also, if you use
tween 20 at 0.1% in 
all hybridization solutions you shouldn't have this
problem with the 
embryos sticking to the columns.

Debby Grant
Research Technician II
Histology Core Facility
Stowers Institute for Medical
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