After transformation in DH5 alpha, I got positive colonies. I have confirmed it by PCR (using an isolated plasmid of positive colonies as a template to run PCR by Takara Taq) and restriction digestion. In PCR, I got exactly the same size of band as my desired interest in the gene but did not get fall out of my gene in restriction digestion.
I have attached a gel pic of PCR and restriction digested . 20 ul of restriction digestion was put at different amount of plasmid ( 5 ul and 8 ul of 140 (C1 )and 305 ng/ul (C2 )
It may be that you are not pcr amplifying the colony but if your primers are insert specific then the wetting of the plate with reaction mix is giving you an amplimer, Ideally in colony pcr one primer should be in the plasmid and the other inside the insert to avoid false positives which make cloning look successful when it may have failed
Thank you Paul Rutland for your answer, but i did not pick colony for colony PCR, Maybe I did not explain my question in proper way, actually i raised all colony which found positive through colony PCR in LB with appropriate antibiotics then only 3 colony grow in broth , then i isolated the plasmid of that colony and then again did PCR with that plasmid, and i got band of same size as my insert , same as positive control.
Yes I may be misunderstanding the problem but if only 3 colonies are antibiotic resistant then it looks like the cloning failed. When you checked the insert was present by pcr of the 3 colonies was your no template control in the pcr negative ( no amplification) or positive
I am also a bit confused as Paul about what you actually did. But looking at your second picture of the digestion, it looks to me that the plasmid was not actually digested. There is no sharp band for the vector. Additionally it may be that you don't have enough DNA loaded to see any 800bp band.
Thank you, Michael J. Benedik. There may be no proper digestion, but I put digestion at 37 degrees for 3 hours, which is the extended time for digestion. Michael J. Benedik , can we send the sample for sequencing for further confirmation because i got results in PCR and have attached a gel picture?
Hello, Renu Kumari I experience the same issues during double digestion. A high concentration of the plasmid will also result in improper digestion. Diluted the plasmid DNA or the volume of DNA adjusted in the Restriction digestion reaction. (Plasmid DNA from colony - 5ul; EcoR1- 1ul; HindIII- 1ul; cut smart buffer- 1.5ul; MilliQ - 6.5ul) . The reaction is incubate at 37 degree for overnight. I will attach the gel image for comparison. Before going for sequencing, you may try once.