I have transformed cloned plasmid in DH5a cells and isolated plasmid from it. when I run gel, expected band should be on 5 kb but it is around 10-11 kb.
I cant sort out the reason for this. Is it possible that plasmid have made dimer? Please give reasons.
Plasmid DNA is circular, it can show a few bands on agaros gel usually called super-coiled (smaller size), linearized (exactly the expected size) and open circular form (big size). These bands vary depend on your plasmid DNA preparation and your gel conditions. in your case, it is more likely you only saw the open circle form in your gel. You can digest the plasmid DNA with a restriction enzyme and then it will show its size of 5kb, if still big, check if it is the right plasmid.
1. Check your target gene by colony PCR or by using purified plasmid as a template.
2. Do restriction digestion to make the circular Plasmid into linear. Because the linear plasmid will give you a correct size of the plasmid.
3. There may be a chance of contamination with other plasmids/vectors.
4. There may be a chance of coupling of two vectors and two insert which forms a single plasmid ( Am not sure). In such case you can't troubleshoot by RE digestion or colony pcr or by sequencing. Simply discard the colony and take a fresh colony from the agar plate.
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