Colony PCR not working - (Mar/18/2012 )
I have been experiencing problem with colony pcr (of BL21) after transformation. I did the mini prep from the same colony and performed pcr with the isolated plasmid (pet with insert) and colony. The pcr was positive for plasmid but did nt get any result for colony. It was strange for me to confirm the insert i performed single restriction of PET with insert and it indicated that insert is present. I repeated the colony pcr 3 times but could nt get result. Can any one help me in this regard
We have no clue what went wrong, since we have no clue as to what you did. Many more details, please.
I prepared same master mix for the plasmid pcr and colony pcr and for +ve and -ve control. I got exact bands for plasmid pcr and in +ve control but in the colony pcr. I transferred a single colony in 25 ul reaction mixture from master mix. PCR conditions in first experiment for all the plasmid and colony pcr (given bleow). Note plasmids were isolated from the same colonies that are not giving colony pcr. in Second attempt I changed the initial 95C from 5 min to 8 min but still there was no result.
95 C 5 min
95 C 1 min
54 C 1.5 min
72 C 1.5 min
72 C 20 min
The most likely problem is far too high a concentration of cells in your colony PCR reaction. Use a 10 ul tip to touch the colony, then use the tip to inoculate 50 ul of sterile water by scraping the tip around on the side of the tube. Use 0.5 ul of the water in your PCR reaction.
thanks i will try this