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Cannot see insert with colony pcr

Colony PCR Cloning

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6 replies to this topic

#1 Smog187

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Posted 02 December 2012 - 05:51 PM

Hello everyone,

Recently I did INfushion cloning of a 4.2kb insert into a 8.1kb plasmid.
I picked 6 colonies and did a colony pcr (using the same primers I used for INfushion) and ran the products on a gel.
So for the gel I did not see any bands at 4.2kb, but I do see multiple bands in each lane with the "highest" band I see at 1.0kb and multiple bands below that.

I'm not sure if this means my colony pcr did not work? or if I didn't successfully clone my insert?

Thanks.

#2 bob1

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Posted 03 December 2012 - 11:20 AM

It sounds like it didn't work. Use the plasmid sequencing primers for confirmation of the insert, it is much more reliable and less likely to pick up unligated insert off the plate.

#3 phage434

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Posted 03 December 2012 - 03:50 PM

Or use one on the plasmid backbone and a second on the insert. Make sure you are using very small numbers of cells. Touch a tip to the colony, scrape in 100 ul of water, mix, use 1 ul as template in your reaction.

#4 Smog187

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Posted 03 December 2012 - 06:10 PM

Thanks for the replies: When I did the colony PCR I used the same primers I used for the INfushion reaction, so "hopefully" the only product being made is the insert.

#5 phage434

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Posted 03 December 2012 - 06:14 PM

The problem with this approach is that sometimes there is sufficient PCR product left over from the ligation and transformation to allow PCR to amplify it from the plate, without the colony having a plasmid containing the insert. The vector primers or (my preference) the vector + insert primers will not amplify that fragment. It will only amplify at the correct length a successful ligation.

#6 Smog187

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Posted 04 December 2012 - 08:13 AM

So I didn't pick a colony directly from the plate. I did a re-streak of my colony, and picked a new colony from that plate.

#7 bob1

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Posted 04 December 2012 - 11:21 AM

In that case, it definitely sounds like the ligation failed.





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