Dear Andrei Nikonov,

Thank you so much for the invaluable suggestions. I invite more comments if your time permits. Thanks a lot.

i) I used BamHI for liniarization of plasmid. it produces sticky end. as you suggested I will try other enz that produces blunt end. To me, it looks like degradation only as I get the same problem with radiolabelled NEB low range RNA marker also(I radiolabelled). In ladder also I am getting far too many bands of somewhat equal intensity. I checked quality of ladder in agarose/ ethidium bromide and looks fine (only 5 bands as expected).  

iv) I will try acidic phenol as per your suggestion.. and also, all solutions fresh. I had taken care of heating problem by constant monitoring and lowering voltage if required... temp never went above 45 degree celsius. I feel, recovery of RNA is fair as I use glycogen for precipitation. Again the villain is RNA degradation!! 

vi) I am getting degradation in control also where no enzymatict/ nuclear extract treatment was done. I must try phenol before i get back to you further.

Thank you sooooo much.

I also recommend using phenol, I used home made phenol/chloroform/isoamylalcohol solutions and never had any problems. Moreover, if your transcription is OK you should not need to purify the MINX RNA (very small and easy to transcribe) . I used it a lot before and found that it was not necessary to purify it. If you precipitate the RNA with ammonium acetate and ethanol  you should minimize the precipitate of free ribonucleotides. 

Good luck

PS: add RNAse inhibitors during transcription and splicing reactions, this really helps. 

The fact that your size ladder is giving too many bands also suggests to me that the RNA might not be completely denatured - I'm guesssing the ladder has not been through the same experimental protocol as your samples. Do you know what temperature the gel is running at? Might help to increase the voltage, and/or prewarm the samples in denaturing buffer before loading.

Thank you Andrei, Isabel, Barabara... I have ordered acidic phenol..hope it will help.

@Barabara- I run 20cmx30cm gel at 700 V.... gel doesnt heat up much... only mild....

And I denature RNA at 80 degree celsius for 3 min and then cooled in ice. I hope phenol should help me....

Thank you all once again.....

Hi Andrei... u exactly said wt is the problem. I changed to new acidic phenol and now works fine... no degradation... thanks a lot.... Isabel and Barabara... thank you so much for your quick reply...

Anyways, now my splicing reaction is not working.. any guess? i think i have to start with a new batch of HeLa nuclear extract?