why no insert but can PCR insert out? - (Jan/25/2007 )

I encounter a strange thing. I inserted a fragment into a vector and get many colonies. I checked 20 colonies with 2 enzyme digestion (NotI&XbaI) used to ligate the fragment into the vector. However, no insert was detected from the gel. Only one big band around the size of the expected linear vector??(it's hard to tell the exact size, as the marker is 12kb, while in my experience, linear 9 kb vector even above the 12kb marker band). But I can PCR the fragment from the colony plasmid DNA (I checked 3 colonies), the size of which was the same as the PCR product of the fragment which was done along side as a positive control.

One person in my lab told me, after insertion, one of the restriction site may not be able to cut again, due to reason like methylation of your fragment near the site, and the big band I saw may be the linear construct with only one site cut. I was suggested to sequence to check whether there was insert because digestion failed to check insert.

Does anyone have the same experience or give me any suggestions? I really feel bad, since I was very optimistic about this experiment before. Thanks.

I agree that you should sequence it and confirm if the insert is present in the construct.
Could it be your positive PCR is from contamination with your insert?? (This may not be true, I am suspecting only. )
Keep optimistic, this is research, therefore, things may not always work exactly as you wish.
Good luck.

what abotu the flanking sites? do you have any? try to digest with different restriction enzymes that are flanking your PCR insert.

What is the estimated size of your recombinant vector?? May be you can do single digestion of the NotI and XbaI to check whether they cut your recombinant vector, along with your empty vector, in addition to the double digestion. If your linearized recombinant vector is bigger than your empty vector, then you may have your insert there. You may use other RE sites in your vector to check also.

For PCR, I may suggest to use other primer set, e.g. sequencing primer M13F and M13R, which flank your insert in the recombinant vector. Or you may use a combination of one sequencing primer and one of your own specific primer, if conditions (Tm, etc...) suitable.

Of course sequencing will give the clear picture of what you have cloned...

Don't worry, many of us encountered the same thing once upon a time, facing it now, or may have it in the future. This is research, re-search, repeat and search...

Hope these would help.

Good luck and happy always..

From your description, it sounds like you used the same primers to check for an insert that you used to create the insert. This leads to false positives when you are checking colonies.

Colony PCR initiated from transformation reactions can give rise to false positives if the primers anneal to the insert sequence due to excess DNA insert from the ligation reaction that is spread as part of the transformation reaction onto bacterial plates. (Qing Dallas-Yang, Guogiang Jiang and Frances M. Sladek, 1998. Avoiding false positives in colony PCR. BioTechniques 24 (4) 580-582) Typical ligation protocols utilize 5 pmol of insert PCR product in a 20 µL reaction (0.25 pmol/µL) with 0.5 pmols of vector. Assuming that all the vectors acquire a single copy of insert, 4.5 pmols (0.225 pmol/µL) of excess insert remain in the ligation reaction. 2 µL of a 1:5 dilution of the ligation reaction (0.09 pmol) is electroporated and diluted 1:9 with SOC media (0.01 pmol/µL). If 100 µL (1.0 pmol) is spread on a 55.4 cm2 plate (0.018 pmol/mm2) and a 4 mm2 agar pick is used, then 0.072 pmols (4.35 e 10 molecules) of background insert are available for a 100 µL colony PCR assay. To avoid this problem, at least one primer should anneal to the vector. Use of the Forward and Reverse Sequencing primers is recommended, as this allows the determination of double inserts or vector-only colonies. A negative control consisting of vector-only is also recommended for purposes of band identification.