non radioactive for southern blot - (Feb/16/2007 )
i have been trying to set up Southern blot using Amersham's nonradioactive kit. But until now i have been unsuccessful in it. i have followed the protocol closely, made changes to salt concentration during the washing steps but i have not been able to detect the bands i require. The positive controls i've used is the PCR product from my clone and also the whole clone, the bands are very clear and good using ECL. But the digested DNA of 10 ug or 30 ug, do not give any results. i have sequenced my probe i and it is correct. i really don't know what i'm doing wrong. could anyone help me to suggest where i could be missing it? i really appreciate it.
we regularly use Amersham's nonradioactive kit for Southern and northern and dot blot hybridization. we never face any problem with the kit.
our protocol is
after electrophoresis we take the picture and then denature the DNA with acid and alkali treatment. then we blotted it in membrane. after blotting we use autolinker and they left for air dry,
in the mean time we label the probe with the above mentioed kit.
we use 1 ul (100 ng/ul DNA) add with 9 ul supplied water and denature it in PCR machine for 95c for 5 min and then kept on ice for 5 min .
then we add 10ul reaction buffer, 2 ul labelling buffer, 10ul crosslinker working solution (20 ul crosslinker +80 mul supplied water)
and incubate it in 37c for 30 min.
in the mean time we pre-hybridized teh membrane with hybridization buffer for atleast 15 min. and then add the labelled probe before adding the probe in the hybridization bottle we dilute the probe with the buffer from this bottle and then add it in the bottle. we DON'T add the labelled probe solution directly to the hybridization buffer.
i hope it will help u
thanks for the reply. i'll try as you have suggested.
i would like to know if you are using it Southern for detection of a single gene copy.
yea, i used it in Southern for detection of a single gene copy