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help - stable transfection clone pick up

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17 replies to this topic

#16 chicken



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Posted 11 April 2011 - 10:13 PM

i also suggest you to scrap ur colonies in microscop with , 20 ul tip,(if you use 100 or 1000ul tip it will pick excess media that will also pick other neighbouring colonies)

so picking colonies with tips is safe.

i first clean microscop with etanol, sterilize it properly, then plac it in hood, and start picking colonies,looking through microscoope.
in start it would be difficult ,but practice makes a man perfect. :)

Edited by chicken, 11 April 2011 - 10:14 PM.

#17 emmanuelle



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Posted 19 July 2011 - 01:07 PM

we add one drop of trypsin on the clone and wait for a while like 1 min. then add 20-30ul media to pipette and suck out

#18 frna11



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Posted 14 August 2011 - 08:01 PM

I've recently selected for clones as well and went with the cloning ring approach as said my zhwong. You do have to be careful with this as the rings can slide across and if they do that your colony will likely get scraped off the bottom and be floating in the media! I always placed cloning ring down and removed media first before adding trypsin to minimise chances of sucking up extra colonies (and to make the trypsin work faster).

As rkay said it is possible that you will have some mixtures in your colonies, but if you see that there are only very few colonies that survived the selection process it is quite unlikely that there will have been two founders close enough to form one indistinguishable colony. The problem with selecting single cells is the extra time it will take to grow your cell line back up to a usable concentration! Also, I'm guessing that you wouldn't use multiple cells from a single colony?

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