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A faster way to pick single colony clones for PCR screening?

single colony clone transformation

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3 replies to this topic

#1 wyyeh

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Posted 23 May 2012 - 04:05 PM

I was wondering if there are any faster ways to perform screening on the transformed comp cells? We usually plate the transformed cells onto agarose plate with amp selection, grow over night, pick and grow 10-20 single colony, and use PCR to screen the colonies with the DNA insert. I find the colony picking step rather slow and tedius as times, especially if I have multiple constructs to screen. Is there any way to speed things up? ~ Thanks!

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#2 phage434

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Posted 23 May 2012 - 05:11 PM

I do it by setting up a plate or strip with 50 ul pure water in each well. Then I use a 10 ul tip on a pipettor to touch a colony. The tip is suspended in the water and scraped against the sides of the well. The same tip is used to deposit 3 ul of the water onto a gridded master plate, then discarded. Afer picking colonies, a multi-channel pipet is used to transfer 0.5 ul of the water into a 10 ul pcr reaction with one primer on the insert and the other on the vector backbone. The master plate is incubated and the correct colonies prepared from the colonies with successful PCRs. If you feel confident, you can grow up some 10 ml cultures from a small number of the picked colonies on the chance that one of them is correct, potentially saving some time.

#3 wyyeh

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Posted 25 May 2012 - 12:27 PM

Thanks!

#4 biznatch

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Posted 28 May 2012 - 09:10 PM

I've done something similiar to the above post.

I set up a gridded plate and a complete PCR mix in uncapped PCR tubes (strips or plates). I use a sterilized pipette tip just held in my clean gloved hand to scrape off the colony, scrape a bit onto the gridded plate, then place in a PCR tube and let it sit there until I've collected all the colonies. An 8x8 gridded plate works well with 8-tube PCR strips. It's very fast, and you'll get enough cells on both the gridded plate for more bacteria to grow and in the PCR mix for a strong PCR reaction.

I originally would just put in in water then add a few uL of the water to the PCR mix, which worked, then to go faster I tried with a PCR master mix minus the Taq (didn't want Taq sitting in the MM for so long) and that worked, so I went ahead with the Taq pre-added and it also worked. I'm not even using hot-start Taq. If I have more than one plate to do I'll split it up so the MM + Taq doesn't sit at RT for more than a few minutes. ie. do one plate's worth of reactions in one PCR block and start it, then set up a new MM for each subsequent plate.

Edited by biznatch, 28 May 2012 - 09:14 PM.






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