So I generated two fragments (A & B ) using PCR and using TA cloning I inserted the fragments into pTZ57R/T plasmids to generate plasmids: ptz-a and ptz-b.I would then, use asc 1 and hind 3 enzymes ( as in attached rough sketch image) to cut out each fragment and then ligate them together to form ptz ab, so digestion of ptz a would be 3220 bp and include the fragment a, while digestion of ptz b would only be 249 bp and include fragment b. so i would ligate the two hind 3 will ligate to hind 3 and asc 1 to asc 1 ultimately putting fragment a and b together ( FIGURE 2). So basically after digestion from ptz b with hind 3 and asc 1 i would extract the 249 bp fragment and from the digestion of ptz a i would use a 3220 bp fragment ligate the two n get ptz ab. however my ptz b plasmid has the insert in the reverse orientation ( figure 3). so my supervisor said I would have to search for new enzymes to cut ptz b . He was saying this because if i had kept to the same plan as before the fragment from ptz b that i would need to ligate to the fragment of ptz b, would now be 3051 bp instead of 249 bp. so ultimately it would mean fusing 3051 bp to 3220 bp. is this possible? or does ligation always need to have a very small insert? or is he saying this because somehow if its in reverse orientation it changes reading frame or something. im so confused.
sorry for the bad image its just a rough sketch
Edited by KhaleesiDany, 25 August 2014 - 03:25 AM.