knockdown issue...please help... - shRNA pGIPZ vector (Feb/03/2010 )
Hi...I need some help..really desperate for an answer..
I produced some stable cell lines with a knockdown of gene...I selected them with Puromycin and cloned the mixed population of cells and performed real time...I tested 27 clones and selected 5 that had a 80 % knockdown . confirmed the knockdown by real time and western blot. This week I made new lysates and prepared new RNA, repeated the real time and western blot. No knockdown. This is unbelievable. I repeated the real time with my RNA from last week and it still has 80 % knockdown. My brain is not working anymore and I am getting crazy 
. I feel like banging my head on the wall 
. I have been working on this since the last couple of months. Last week I thought it worked and this week , this wierd thing, Does anyone have any clue what is going on?
Please Please Please help....
Priya914 on Feb 4 2010, 04:45 AM said:
I produced some stable cell lines with a knockdown of gene...I selected them with Puromycin and cloned the mixed population of cells and performed real time...I tested 27 clones and selected 5 that had a 80 % knockdown . confirmed the knockdown by real time and western blot. This week I made new lysates and prepared new RNA, repeated the real time and western blot. No knockdown. This is unbelievable. I repeated the real time with my RNA from last week and it still has 80 % knockdown. My brain is not working anymore and I am getting crazy
. I feel like banging my head on the wall . I have been working on this since the last couple of months. Last week I thought it worked and this week , this wierd thing, Does anyone have any clue what is going on?Please Please Please help....
not using RNAi myself but what i think is that you lost you construct in the process retaining the resistance (if you're still using selectiion). you should check if your cells still have the complete construct
Fiz