Hello Muatasem

 I suggest to care the following 2 points for in situ experiments.
 In situ people may suggest to pay special care to contamination due to RNases. However too much care is not good, because it make hesitate for peoples to take in situ experiments.
In my experiences, I have had no failure due to RNases, I believe it may be more important to routinely see RNA recovery using formaldehyde-containing agarose gels after RNA syntheses. Very good riboprobes are the key to success at in situ experiments.

As in in situ hybridization (Loomis and Escalante), it is important for RNA to be dissolved in H2O, because peoples have customs to dissolve nucleic acids in TE buffer,  but TE contains EDTA, it inhibits T3 enzyme. Also, T3 and SP3 enzyme activities are inferior than T7, it is important use more substrates (DNA) in these cases.
good luck,

Hiroshi Ochiai

--- "Ubeidat, Muatasem" <[log in to unmask]> ---

>Hello Dicty People,
> 
>We are also doing in situ hybridization according to Loomis and Escalante. If you have suggestions and improvements of the procedure, please send us your valuable comments. Thanks. 
> 
>Muatasem 
> 
>MUATASEM UBEIDAT, Ph.D.
>Associate Professor of Genetics and Molecular Biology
>Southwestern Oklahoma State University
>Department of Biological Sciences
>100 Campus Drive
>Weatherford, OK 73096
>Tel (580) 774-3298 (Office/Lab)
>Fax (580) 774-7140
>E-mail: [log in to unmask]


Hiroshi Ochiai,
Emeritus Professor
Division of Genomedynamics
Creative Research Initiative SOUSEI
Hokkaido University
North 10, West 8, Kita-ku,
Sapporo 060-0810,  Japan
Tel: 011-706-3588