Originally Posted by TuanVo
I study about cloning. My gene are too long (about 5,200 bp). I use Phusion DNA polymerase to amplify this gene but I got many wrong bands and right band was very thin. I would like to reduce wrong bands and increase my target band. Please help me.
Thank you very much
Try purifying the 5.2kb band and using that as template for PCR with the same primers (i.e. re-amplify it).
If your initial template is cDNA this usually helps because it greatly reduced the complexity of your template sample. However, if you're amplifying from plasmid I don't think this will make a big difference, in that case start playing with the PCR conditions.
Another way to do it is to split the gene into two halves, PCR the halves independently and then stitch them back together again.