I am working on cloning of a gene fragment inside my vector everything we tried to do correctly like making insert and vector with utmost care but after ligation the morphology of colonies seems to be very unusual i m wondering why this is happening. Currently i am using TOP-10 competent cells for cloning and antibiotic which i am using for selection purpose is ampicillin any suggestion will be valuable to tackle the problem thanks
B R Siddharth Are you using commercially, ready to use top-10 competent cells?
Check the genotype of host strain (top-10) for antibiotic resistance.
Use freshly prepared media for transformation procedure. Try with Dh5 alpha or JM109 host. Also check for antibiotic resistance marker in vector. Are you using correct concentration of ampicillin for selection colonies?
These don't look like Top10. Top10 around 16h past transformation ("over night") form tiny colonies when compared to BL21, about 1mm diameter. Maybe looks like that if you had them in the incubator for one more day or over the weekend at room temperature, never tried that with Top10.
During cloning, there is little reason not to utilize Golden-Gate cloning to at least some extend. It relieves you of the whole gel-separation and extraction duty which will waste 80% of your DNA, with rather varying purity of your vector DNA.
If your vector doesn't have the desired type II restriction sites, especially the vector backbone requires diligent digestion and separation, as it will lead to contaminate colonies.
I prefer to cut my non type II vector from a GFP expressing plasmid, as contaminate full vector colonies can be easily discerned under UV via promoter leakage (you could also use the commercially available CcdB plasmids, which require a special host strain like DB3 to be amplified).
Lately I forgot my vector digestion for some 6h at 37°C, judging from the gel it turned out well though, should be rather thouroughly digested. So I guess it's better to overdigest than to underdigest in case of the vector, where you'll have a hard time to separate the undigested/digested vectors. Also, use multiple amounts of insert compared to your vector in a stoichometrical sense. Excess of latter one will not result in contamination. Usually I use no more than 2µL of 10nM vector.
It does look a bit unusual, maybe as if there is some lysis at the edges. You could try a different bacterial host, sometimes that will fix it. But you can also just screen some of these to see if they are correct clones.
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