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Colony-PCR workaround


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#1 Spright

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Posted 22 January 2013 - 12:08 PM

Dear all,

I ligated a 1,400-bp insert into a 3,300-bp vector and transformed this into DH5alpha cells. I obtained colonies, did a colony PCR with the primers originally used to obtain the insert, and I'm getting nothing. This isn't surprising to me because obtaining the insert to begin with had been a big challenge.

My second option, I believe, is to use vector-specific primers for my colony PCR.

Are there other options? For example, I can grow all the colonies, miniprep them, run the extract on a gel and recognise positive according to size. Would that work? Isn't it actually the safest method anyway, since it avoids false positives? Colony PCR, as we know, also amplifies ligation DNA that wasn't absorbed by the cells during transformation, right?

Could I actually skip the miniprep? Could I scrape colonies, boil them for 10 minutes in order to lyse the bacteria and then run the lysate on a gel? I would get, of course, a huge genomic DNA smear, but my 4,700-bp vector would be visible, wouldn't it?

Thanks!

#2 bob1

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Posted 22 January 2013 - 12:16 PM

The mini-prep option is probably the best of them, it also means that you can cut out the insert with REs to confirm the presence as well as going on size.

#3 jerryshelly1

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Posted 23 January 2013 - 06:05 PM

I can't tell you how many problems I have had with colony PCR in the past. Sometimes it works wonders and others, I get false-positives for every sample. I have tried to find time and money savers when it comes to verifying a correct ligation product. The quickest and cheapest is isolating a colony, grow until turbid, pellet, obtain small sample from pellet and perform PCR screening on this. I have had better luck with this and if the PCR confirms the product, you can process that pellet via miniprep and double confirm by digestion.

You are right. You should always use a vector and insert specific primer for PCR screening. Double check your original insert primers. You may not be obtaining a PCR product because the primer RE site overhangs are impeding proper binding to your ligated insert. I only say this because most restriction enzymes require a 1-4bp overhang for proper activity.

#4 Trof

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Posted 24 January 2013 - 12:01 AM

The best worked for me a primer pair with one on the vector and one in the insert (should not get a false positive from a ligation mixture) and always using roughly the same abundant positive control (by band intensity check, you can discard those less positive as false). Also negative control, of course.

There was this protocol my colleague used, it's called Cracking of colonies (this looks kind of similar to our protocol), you can isolate DNA within an hour of gel preparation. But I don't have much good experience for that in case the vector is small. There can be sevaral bands then on gel as the plasmid has more coformations and sometimes some of them are missing in the sample, so It's hard to tell what's the correct vector with insert. But maybe reliable enough for bigger inserts, he was creating constructs, I was merely cloning PCR producs or doing mutagenesis.

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