- PCR with specific primers for the gene with these plasmids width different dilution 1:10,1:20,1:50,1:100 but there is still no band.
- Cut plasmids with NheI and HindIII ( enzymes have recognition site on forward and reverse primers) but only hindIII can cut plasmids, I have checked the activity of NheI and it is ok.
- Cut plasmid with NdeI which have recognition site on L1 gene ( in the middle of the gene and it doesn't have recognition site on pBluescript) and this enzyme can cut the plasmid.
So, after tests above, i cut plasmids with hindIII and BamHI ( I think that they cover the ligated DNA) and I see that there are a 1kb band and a 3kb band ( size of cut pBluescript). In conclusion, with all the results above, I think that my pcr product was degraded and I lose 600 kb at 5'end of the gene during ligation and transformation reaction because NheI recognition site locate on foward primer.
Idont know why this phenomenom happened. Please give me some explainations and sugesstions for my case, thank you very much !
Edited by Awnman, 18 February 2013 - 07:32 PM.