False positive for colony PCR - (Oct/10/2005 )

hi all,

help me in this situation,

after transformed, i got many colonies on transformation plates. not wanting to miniprep all of those colonies, and digesting them, i screened the colonies with doing PCR colony first.(using the primer for the insert)

some of them gave positive band (exact size of PCR product). then, i cultured all the positive product to isolate the putative plasmids and after that digesting them with the enzyme i used to ligate vector and insert.(i'm doing cohesive-end cloning).

unfortunately, when ran on gel, what seem to appear were just the band of insert.

this is so confusing. where goes the vector?how can the insert alone can appear as colonies on transformation plate?help me!! i'm desperately in need of finishing my cloning work..

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.

In the same vein, routine practice in our laboratory is to first pick colonies from the transformation plate to a fresh plate, allow them to grow overnight, and perform colony PCR on them...