Ligation help needed! - vector religation? (Nov/10/2005 )
I am trying to clone a 150bp fragment into a vector using EcoRI and BamHI. I keep getting lots of colonies on my negative control ligation plate (vector with no insert) and few or no colonies for my vector + insert ligation. I know my digestion of the vector worked for both enzymes. I wouldn't think the Eco and Bam cut ends could religate. And if for some weird reason this is happening, why does the presence of the insert inhibit this? Please , please let me know if you have any idea what could be the problem here. Thank you.
There is usually a background consisting of single-cut molecules in the double-cut vector prep. This is why it is a good idea to treat the vector with SAP even when most of the vector is double-cut with incompatible ends.
I agrre with tfitzwater you should treat your digested vector with SAP. It reduces a lot, lot lot the background.
But just in case after that you still have a problem, how was the 150 bp fragment made? Did you cut from a plasmid or did you PCR? If you have done PCR check if you have enough extra base pairs before restriction site. that is vry important to ensure the enzyme can cut the insert.
thank you very much!