I am using a TOPO-TA cloning kit from Thermo Fisher (K4575J10) for cloning. Before the cloning reaction I prepared my PCR fragment and the proper size was confirmed on gel to 400 bp. The cloning reaction and transformation is performed according to manufacturer's manual. After plating and ON incubation, single colonies is transferred to a small amount (10 ul) of growth medium and an aliquot is thereafter used for PCR using M13 fwd and M13 rev primers that is included in the kit. The expected band is around 600 bp, but a distinct 1500 pb is the result from the PCR.
I produced my PCR fragment using Taq-polymerase in one reaction and a High Fidelity polymerase in another where A-overhangs is added in post-amplification, also acording to manual protocol. I got the same result in each preparation and in several clones.
Any suggestions what the PCR-product might contain?
Thank you for help.
Manual (vector map on p.35) :
https://tools.thermofisher.com/content/sfs/manuals/topotaseq_man.pdf
Try enzyme digestion instead of PCR. After you get the colonies, grow them in 5-10 ml liquid media and extract DNA (quick and dirty). Digest using different combination of enzymes (some cutting the vector and some the insert); run on gel to see if you have expected bands. Sometimes, the insert can also ligate in opposite orientation (probably because of A overhangs) and shows correct length when cut at only one site. So use at least 2-3 enzyme combination.
After confirming correct insert length and orientation, send them for sequencing.
I hope it helps.
Good luck.