i used 2 primers to amplify a 2kb region in proviral genome which is integrate to carriers. at first i did this in a virus-transfromed cell line, sequenced the product and use it as the reference sequence. then i used the same condition to amplify this region in carriers. it is very strange that i got 4 bands for every carrier, instead of 1 in cell line. the size ranged from 0.8kb to 4kb. at first, i thought those might be nonspecific bands so i just cut out the 2kb band and cloned it into TOPO vector and sequenced it. i got the right sequence for the first 2 carriers i sequenced. then i optimized the insert/vector ratio in the ligation and i found i could use much less pcr product and get not less clones than before. however, when i sequence these clones, i got no result except from vector-based primers. and more surprisingly, the sequence is totally different from proviral genome that i had sequenced in the first 2 carriers. i blasted it and found it has 100% homology with a region in chromosome 13q. i dont understand. the only difference in method is the insert/vector ratio. is that the reason? or its the problem of different carriers? why cant i find even the primer sequence?
i dont konw how did this happen. trying to find some possible reason, i sequence the other 3 bands and found they all localized in chromosome. it is very strange because when i use the primer sequence to do a homolog search in the sequence, i got no homolog at all. what are these fragments? its driving me crazy, please help me. thanks.
i attached the photo, 2nd band from top is what i need, 2kb.
Edited by gyma, 30 March 2010 - 04:26 AM.