I am trying to clone a 3kb insert into a 5.3kb vector. The colony PCR was positive for around 12 colonies but upon digestion of the plasmid isolated from those colonies, no insert was observed. Only the band of digested vector was observed. How should I troubleshoot this problem?
Hi Malabika,
The and for the digested plasmid is 5,3 kb or 8,3kb?
If it is 5'3 your insert was not cloned and you might have a false positive in your colony PCR.
If it is 8,3 the insert is cloned but not released, then you should check the restriction enzymes you used are working fine (e.g. by digesting another known DNA) or verify whether the restriction sites used for the cloning were compatible in which case both sites are destroyed (e.g. BamHI/Bglll, SalI/XhoI, SpeI/Xbal/NheI, etc).
Hope it helps.
Hi Malabika,
The and for the digested plasmid is 5,3 kb or 8,3kb?
If it is 5'3 your insert was not cloned and you might have a false positive in your colony PCR.
If it is 8,3 the insert is cloned but not released, then you should check the restriction enzymes you used are working fine (e.g. by digesting another known DNA) or verify whether the restriction sites used for the cloning were compatible in which case both sites are destroyed (e.g. BamHI/Bglll, SalI/XhoI, SpeI/Xbal/NheI, etc).
Hope it helps.
Hi Malabika, for colony PCR you may use-
Forward primer: Gene specific
Reverse primer: Vector specific
Thus you can make sure if your insert is successfully cloned into the vector or not. Best wishes.
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