Obtaining linear ssDNA

[superbeamer]

Hi,

I'm trying to obtain linear ssDNA between 5-10 kb long. I have tried cleaving M13mp18 plasmid (circular, 7 kb) with Swa I endonuclease after annealing a 30 bp primer over the restriction site, but I have not been successful in obtaining my desired product. Does anyone know why this might be? I have already tried various optimization steps by varying enzyme concentration, incubation time, etc. Is there another enzyme that might be better?

I have begun looking into other techniques to obtain my final product, such as PCR and separating a double-stranded linear DNA, but I am unclear as to the best approach. Any help would be greatly appreciated.

Thanks!

[BioBM]

Sorry if this sounds obvious, but have you tried a different restriction enzyme and had the same problem?

[superbeamer]

Yes, I've tried the same procedure with Hinc II. I have found examples where this scheme of binding oligos to ssDNA and then cleaving has been used successfully, but for one reason or another it is not working for me. Right now I am interested in trying to obtain the desired product in a new way. Any ideas?

[user10]

Hi,

as you described, you tried to cut the ssdna of m13mp18. That's why I'm trying to contact you. I have tried to cut ssdna by anneling an oligodeoxynucleotide to the restriction site of several enzymes. Till now i wasn't able to achieve an efficient digestion. Only with EcoRI I had little succes. Did you try further experiments and have you been succesful? I would appreciate it if you could give me some tips, i.e. concentration of enzyme, oligodeoxynucleotide and ssdna as well as the type of enzymes you used. I'm looking forward to your respond.

Thanks very much