I was trying to insert the interested gene (2 kb) to the vector (6 kb). Firstly, I used EcoRI and XhoI to cut the plasmid, and treated with CIAP. Secondly, ligate the insert and vector by using T4 ligase. After purifying the DNA from colonies amplification, I used EcoRI and XhoI to cut it. I was supposed to see a band at 2 kb, a band at 6kb, a band at 8kb. But there is a band at 4 kb instead of 6 kb. Can somebody help me out with this problem? Please see the picture attached below,the 3rd and the 4th lanes are the 2 kb ,4 kb, 8kb bands.
Hi,
Don't worry about the exact size of bands, because restrictase buffers usually modified the apparent size of DNA - certainly cause of salts (this is not true if you also put restrictase buffer with your marker).
I would suspect that lanes 2, 5 and 6 correspond to empty vector, but that lanes 3 and 4 are contaminants. If they would have been plasmid with insert, you should expect at leat as intense band at 2kb as at 6kb - which is even not correct as there is more DNA probe in the longest DNA fragments.
So, if I were you I would screen more colonies and if you would still unsuccesfull getting positive colonies, the best is to redo your experiments.
You did not mention the origin of your insert: PCR amplicon or restricted fragment?
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