Insert alone gives colonies?

Malay curiouser at ccmb.ap.nic.in
Tue Aug 22 08:21:02 EST 2000


> No User <no.user at anon.xg.nu> wrote:
> : Anybody ever seen something like this before? On a
> : ligation of a long PCR fragment, much troubleshooting
> : has shown that colonies come up on an insert alone +
> : ligase control plate.  (Minipreps on colonies from the
> : vector + insert plate are either vector without insert
> : (rarely) or (mostly) some small garbage plasmid,
> : smaller than the starting vector.  Also, a vector +
> : insert without ligase control plate gives few or no
> : colonies, suggesting that the colonies on the insert +
> : ligase plate arise through ligation.)

Give some numbers. How many colonies? Did you try isolating plasmids from
the isert only plates? What do you mean by garbage plasmid?

> It is most likely contamination of the PCR product with a linearised
> plasmid -- perhaps the template itself -- that you co-isolated in the
> gel slice.

Possible but rare to see.

>Electrophoresis never gives perfect resolution, and
> virtually any slice you take from a lane will contain at least a trace
> representation of all the DNA you loaded (or in addition, if you didn't
> clean out the tank carefully, a representation of DNAs loaded by your
> colleagues in the recent past!).  That is why two or three rounds of gel
> purification is often necessary.

Again possible but 3/4 rounds of purification is not necessary.

I guess your competent cells are not good and contaminated. Try plating the
cells alone in a proper Amp plate. See that you get no clones there.

Malay
Malay Kumar Basu
Centre for Cellular and Molecular Biology
Hyderabad 500007
I N D I A

Fax: (00-91)40-7171195
Phone: (00-91)40-7172241
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curiouser at ccmb.ap.nic.in





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