Dear all,

 

I am a master student and i just began with siRNA transfections. I have some problems with the scramble. My scramble always give dead cells, so the results couldn't be used. Then only thing that I do differently from what i ve been demonstrated was that i place first the siRNA/scramble drop and after the siRNA buffer in the eppendorf. But that can't be the case, can be? Could you please help? I am trying to find what is going wrong. Only once it worked and i had alive cells in the scramble. I work with a 12-well plate and only mine scramble gives dead cells. Any ideas?

The scramble is just a negative control and should have no effect on viability (usually). What is more likely the case is that there are issues with cell handling. Typical issues include working too slow, getting contamination into the culture (either during handling or due to contaminated chemicals, including water), too much agitation etc.

 

I would check with other lab members whether they have any issues and ask a senior member to look over your shoulders while you do the experiment to see whether they can spot any errors during handling.

Thank you for your help