Hi Sahil,

For polymerization there can be some problem with the gel components. Try to use the fresh components.

Regarding isolation of the radiolabelled band. The simplest procedure will be after you develop an autoradiogram, align it with gel on a white light box (keep the film on the lower side). Then excise the region you need using a sterile blade. Keep the setup behind the acrylic sheet to protect your self from the radiation

bye

Hi. Ajay...thanks for the answer.actually for visualising the bands.i normally dry the gel using gel dryer and leave it the x ray film overnight at -80 degree and then develop. So i cannot use the same gel

i agree with Ajay,

dont dry the gel just cover with clingfilm . you can put 5 or more little spots of radioactive probe in blue dye on the gel just before putting the film on top of it and possibly a couple of nicks cut in the gel and film  to provide orientation of the autoradiograph. you do not need -80 development ..the film will develop about 4-8 times slower at room temperature compared with -80 and depending on the thickness of the gel but it should give you a signal and you do not need a strong large signal which hides the exact position of the band just a weak signal will be enough

Hi Paul... Thanks a lot.I will definitely try this procedure.

Hi Sahil,

Prof Rautland has further elaborated the procedure for perfect alignment of the gel and the film. I used to follow the same procedure and it is simple and works...

bye and all the best

Hi paul, How long will i keep  the x ray film on gel to develop at room temperature.

i cannot say but a guide will be to assess how long you leave the dried gels and whether you get a really strong signal and if you think the signal is 10 times stronger than it needs to be for easy visualisation then put the room temperature gel down for about half of that time but i would put the first wet gel on film for 1 hour at room temperature with an intensifying screen and then put it down again if necessary depending on the strength of the signal . you do not need a strong signal just enough to be sure that it is the correct band

good luck

paul

Thanks paul..i tried..it worked..thanks for your assistance :)

Well done Sahil  I am so glad it worked. I hope the project goes well.all the best

paul