problems with PCR confirmation of insert . HELP ! - (Aug/22/2011 )
i have to make clones of viral genes l And p . i have made the c dna and amplified with PCR and purified it . then i inserted it in T easy vector and transformed in the host DH5 alpha cells. and i got clones too . in order to confirm the inser i did restriction digestion and i got the exact band desired . further when i did PCR for the particular gene the band is coming below the band for the desired one.its the same for other clones . i am clueless of wat is going on . can anyone help please ?
Your primers could be binding to sites other than the ones you intend. There could be repeats in your gene, or incorrect priming due to too low an annealing temperature. I would sequence your construct.