Hi!

Unfortunately working with M13 is not trivial. The appearance of shorter M13 phages (with relative shorter DNA then the original) is known to happen due to recombination. You should consider to do not let the culture grow for too long and to do not refresh it many times. 

How are you purifying the ssDNA? With a commercial kit?

I would suggest: http://www.qiagen.com/products/catalog/sample-technologies/dna-sample-technologies/plasmid-dna/qiaprep-spin-m13-kit

For my experience it is always best to gel purify the plasmid anyway.

Good Luck!

Diego

Hi Diego,

Thank you very much for your suggestions that are very helpful to me.

To purify ssDNA I use a published protocol, which is performed with a step of protein stripping and a second step of DNA purification by ethanol precipitation: http://www.nature.com/nmeth/journal/v8/n3/full/nmeth.1570.html

ssDNA at high yield ( ~0.8 mg) and high "monodisperity" have already been produced in our lab. with the aforementioned protocol. The last time that I produced ssDNA I started to have troubles with purity. I repeated the experiment from the beginning 3 times getting always the same results.

So, I going to change protocol following your suggestions. Of course, I would like to update you with my results.

Thanks,

Alex

Hi Alex,

Have you solved your problem? 

I'm also working on the ssDNA recently.  Could you please tell me how to run the Agarose gel for ssDNA? I could only get very faint bands every time using the electrophoresis protocol for dsDNA.

Thanks a lot,

Best,

Xiaobing

Hey Xiaobing,

try to use a loading dye which includes SDS or simply add it yourself to your dye of choice. 1% in a 6x stock should do the trick.

Bes,

Andreas

Hi Xiaobing,

sorry for late response!