EMSA problem

[rmohan]

Hello

I have performed EMSA and could see the shift after SYBR green staining.... but unforunately I could not see the shift for the same sample when I am using the radiolabelled probe.

only difference i could sense is the DNA conc. , in radiolabelled probe ( DNA conc. very less) compare to DNA what I have used for the SYBR green staining ....

why I am not observing the shift when i use radiolabelled probe !

please give me your suggestions...

thanks in advance and waiting for ur suggestions...

[admin]

Hi Rmohan,
i can understand your frustrations. What probe did you use: P-32 or other? What nucleotide did you label? Is the probe made the exact same way in both experiments (salt, buffer, conditions)? Have you tried non-radiolabeled probe?

[rmohan]

hello

thanks

I have labelled the probe ( PCR fragment ) with end labelling (T4 kinase- gamma P32) and purified.

Iam sure probe is fine on gel with intact band , same PCR fragment used for the experiment and stain with ETBR or SYBR I could observed the shift , but not in the radiolabelled probe !... same conditions for the both the expts , no change at all.

I have not tried with non radiolabelled probe ( I mean not DIG labeling).

lookinig forward for the suggestions....

[andyzhoucang]

hi rmohan,
can you post you image?