Replacing small gene with large gene - (Feb/23/2007 )
I am trying to clone a 2700 bp gene in a 4 kb vector. I am digesting vector with BamHI and AvrII (releasing ~20 bases) and trying to clone the 2700 bp gene (digested with BglII and XbaI). I have tried a couple of times using various combinations of gene cleaned vector and insert part but could not get any colonies after transformation even in the negative controls (gene cleaned vector without ligase and gene cleaned insert without ligase). Any suggestions??? and thanks in advance
Clone the gene as two fragments, either clone in piece by piece or perform a 3-way ligation. Smaller inserts clone more efficiently.
how much DNA do you use for ligation, what ratios. Increasing DNA amounts and higher ratios can help.
Check your ligase buffer and ligase. They tend to degrade over period of time.
Could you also verify the cells for transfomation are also fine.
One wouldnt expect to find colonies in negative controls. so thats good. But is it possible that you overdigested the ends which could also give a similar result.
Good Luck !!!