Protocol Online logo
Top : Forum Archives: : Molecular Biology

Screening stable clones - (May/22/2007 )

Hi!

Need some help.

I'm stable co-transfecting diferent cell lines, meaning that I have my resistance gene in one plasmid and the reporter (luciferase) in another. Whenever I start picking resistant clones, I hope to have several 96 well plates with them awaiting to be screened for reporter response. How do I screen them? I can't have all those clones in the 96 well plates grow into confluent 12 well plates and start screening them with a standard luciferase assay. That doesn't seam practical. How do you guys do it?
I wonder if this is a good idea:
let the clone polulation on the 96 well plate grow to confluency, harvest and split in 2. One to continue culture and the other to screen by PCR.

What do you guys think?


Thanks in advance.

-dub scientist-

For each set of stable clones, we pick 16 and transfer to 96 well. And from this we transfer to 24 well plate and we find that only some of the 16 actually grow confluent. From the 16, we select the 6 most confluent ones and grow them in 6 well plates and then analyze them by wetsern blot.

-scolix-

QUOTE (dub scientist @ May 22 2007, 10:00 PM)
Hi!

Need some help.

I'm stable co-transfecting diferent cell lines, meaning that I have my resistance gene in one plasmid and the reporter (luciferase) in another. Whenever I start picking resistant clones, I hope to have several 96 well plates with them awaiting to be screened for reporter response. How do I screen them? I can't have all those clones in the 96 well plates grow into confluent 12 well plates and start screening them with a standard luciferase assay. That doesn't seam practical. How do you guys do it?
I wonder if this is a good idea:
let the clone polulation on the 96 well plate grow to confluency, harvest and split in 2. One to continue culture and the other to screen by PCR.

What do you guys think?


Thanks in advance.


idea is good; but there should only be a single cell per well of 96 plate to ensure cloning; I think there is no need to screen several 96 plates beside if you like to isolate several dozens of positive (stable) transfected clones (but why?)

-The Bearer-