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bob1, on 28 July 2012 - 04:08 PM, said:
How did you purify the plasmid post picking the colonies? If you did a miniprep, you may have had some residual medium left behind, which means that you get some bacterial decay products (cell wall etc) left behind, that inhibit digests post purification.
It could be that the colonies have more than one form of the plasmid in there.
What controls did you have for the ligation?
It could be that the colonies have more than one form of the plasmid in there.
What controls did you have for the ligation?
I should also add, I tried to do a transformation with the plasmid purified, that failed too!
insert, no plasmid, ligase, C) insert, no plasmid, no ligase, D) plasmid, no insert, ligase, E) plasmid, no insert, no ligase, F) no plasmid, no insert, ligase and G) Pet15B without digestion.
