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Blue colonies with insert, white colonies with multiple weak bands

Topo TA cloning blue colonies

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#1 Placebo

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Posted 02 March 2012 - 04:47 AM

Hello,
I am new to cloning, and in my lab there is not much expertise. I did my first cloning a couple of weeks ago (Invitrogen TOPO TA), inserting a (very short) fragment of 150 bp, and it seemed to work well. But there are several problems:

- The white colonies give multiple weak bands in agarose gels after PCR with M13 primers; one of the correct size, as well as other larger fragments.
- The blue colonies also seem to have the insert.
- The PCRs from the blue colonies give a FAR STRONGER band, without any extra bands.
- Sp6/T7 primers give only an extremely faint band or none at all
- The above happens both in colony PCRs and PCR after plasmid extraction

What is going on here? What is the secret behind the excellent band from the blue colonies?
Sincerely,
Jon

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#2 bob1

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Posted 02 March 2012 - 12:44 PM

The bands you are seeing in the PCR are the result of primer-dimers. You will probably be better off screening one of two ways - either digest out the band using restriction enzymes (if possible), or having one primer anchored in the insert - this will give you a short product, which may migrate below the primer dimers, but there should be no false positives. Increase the % gel to get better resolution of the small prosucts

#3 Placebo

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Posted 05 March 2012 - 06:56 AM

The bands you are seeing in the PCR are the result of primer-dimers. You will probably be better off screening one of two ways - either digest out the band using restriction enzymes (if possible), or having one primer anchored in the insert - this will give you a short product, which may migrate below the primer dimers, but there should be no false positives. Increase the % gel to get better resolution of the small prosucts


Correct me if I'm wrong, but aren't the primer dimers way down at 50-60 bp? The insert is larger than that, and I have already tried using the primers for the insert alone. It works, but gives a very weak product and also two or three extra bands. Again, the blue colonies have the insert, and mysteriously give a much stronger band. I will probably try a different kit.

#4 Placebo

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Posted 08 March 2012 - 01:00 AM

It seems we have a clue; the ampicillin we used seem to have gone bad. Perhaps it was killed by light, as it was stored directly under the little light in the freezer. Several years of short, daily light exposures may have destroyed it. The plates we made with the old ampicillin were totally covered by a dense mat of bacteria, while the plates with new ampicillin had normal colony densities. If I am correct, only the blue colonies actually had plasmids, and by chance those that I tested also had the insert.

#5 bob1

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Posted 08 March 2012 - 11:57 AM

If you are screening by picking directly off the plate, it is relatively common to pick up untransformed products from the plate, such that they give you false positives.

#6 Placebo

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Posted 09 March 2012 - 09:41 AM

If you are screening by picking directly off the plate, it is relatively common to pick up untransformed products from the plate, such that they give you false positives.


That's probably it. It seems all our problems are solved; I now get VERY nice bands of the correct sizes. The blue colonies from the previous cloning did indeed contain the correct insert ( we sequenced them).





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