We used to ko b2m by talen. 1, two copies of b2m are same in your T cell (maybe you have two different copies in wt) ? 2, you have the positive and negtive control in pcr?

Protein level check is more important (Wb and FCM). So I think your knock out is work. Good luck.

Hi Kristina:

To assess intracellular protein expression amount by FACS has to be cautious, escpeically there is only some shift, instead of complete resolved population. Off target or indirect effect may induce FcR expression change, which will shift the histogram peak more or less.

Validation with WB or Confocal should be helpful.

Best wishes

Shiqiu

Do you grow the cells from a single cell colony after KO? Heterogeneous KO will result in mix of KO, "partial" KO (only one copy KO) and Non-KO cells. So, you can have the sequences differs. Using haploid cell lines will reduce possibilities of more copies, but I don't think you can easily change the cells. Anyway, good luck!