We obtained M13 ssDNA from NEB and transformed suitable host bacterial strain followed by phage preparation from the same. Phage was then used for purification of ssDNA by PEG precipitation and phenol extraction (detailed protocol was followed as given in Molecular Cloning by Sambrook and Russell) and was used as a substrate for enzyme assay. There was no activity found. However, when M13 ssDNA supplied by NEB was used for the assay, enzyme activity was detected. When both DNA samples (isolated in lab and supplied by NEB) were run on the gel, one isolated in lab was found to run slower than that from NEB (image attached). Is there something wrong with the way I am isolating ssDNA even though M13 phage used for this was prepared from ssDNA supplied by NEB?
Hi Ankita, as shown in your fig sharing had been occurred for your DNA during preparation that implies inability of enzyme to digest your preparation. But in some cases when DNA not purified well or increase salt concentration in your preparation my lead to this condition like sharing. So my advice is to detect A260/280 ratio to use suitable concentration of purified DNA and remove excess of salt conc. Then try to make running again unless your DNA may be damaged during preparation.
regards
Thank you for the suggestion but enzyme that I use for assay is not a restriction enzyme. It is supposed to synthesize a product using ssDNA as a substrate.
sorry, for mistake and thank you for correction but in both cases ss DNA acts as a substrate.
regards
Your samples look like genomic DNA to me. What host strain did you use?
Well, that strain is perfectly fine for your purposes. Why don't you try a test digestion of your samples with Sau3A I (or any other enzyme with a 4 bp recognition site)? If they are digested, then at least you know that what you have is genomic dsDNA and can start troubleshooting from there.
Another thought. Have you checked the TcR marker of XL-1B MRF'? That would let you rule out a mixup with an F- strain....
ssDNA from phage is circular but can be broken in the extraction process and migration in agarose gel is slower.
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