4 replies to this topic

[seashell83]

I am going to do stable knockdowns for a gene of interest and this is the first time I'll be doing that. My question is I want to check 5 target sets from the company Sigma for my gene of interest and with each target  I will have to check which one knocks down the gene better. Since the beginning experiments will be in a 96 well to 24 well plate , will I have enough cells to perform westerns? What would be a suggested method to look for best knockdown by the  target set.

[pcrman]

I guess shRNAs sold by Sigma are validated. 24well barely give you enough cells for Western. I would say 96 - 24 well plate is good for qPCR validation.

[Functional Screens]

The shRNA clones/vectors sold by Sigma might be from TRC.  I don't think they are validated.  I will say there is no one company in the world selling shRNA/siRNA collection which are ALL validated.

Reporter asaay could be an option to screen potent shRNA in 96-well before making stable lines.  Also, the reporter asaay can be carried out in 293T cells, the most transfectable cell lines, in case your model cells are not very transfectable.

Hope it helps.

[dp5]

Sigma does carry TRC clones.  Not all are validated, but many are.  Validation data is viewable when you log into the website.

[warsel]

dp5, on Jun 18 2009, 08:17 AM, said:

Sigma does carry TRC clones.  Not all are validated, but many are.  Validation data is viewable when you log into the website.

I work with a big set of TRCN vectors as well and validated them in 96W on western.
You don't need more lysate than you can load on a gel anyway and a confluent 96W lysed with 50uL sample buffer is really more than you need.