I am new to cloning ...and I have a question to ask...I am cloning a gene inside a low copy number plasmid...and in the MCS the two restriction enzymes sites have only 6bp difference hence are close by....I performed a double digestion of this plasmid and insert and got colonies after ligation...with 2-3 colonies on vector control plate...I ran the plasmid prepared from these colonies to compare that of the untransformed plasmid..and instead of the transformed plasmid running behind in the agarose gel than the untransformed..i observed that the transformed plasmid ran a little ahead of the untransformed plasmid....on restriction digestion no insert was obtained but plasmid was seen on the gel....I have two questions
1. Is one of the enzymes not working and so religation of vector is taking place cos colonies are there after transformation..
2. If two restriction enzymes sites are present at 6 bp difference is there a possibility that double digestion will not work? do i have to redesign the primers??
3. Is alkaline phosphatase treatment necesssary for ligation in this case...
Thanks in advance...any help will be appreciated
Edited by vinee2, 01 March 2012 - 08:27 AM.