subcloning dirty pcr products

Emma Macfarlane emma at HERA.MED.UTORONTO.CA
Fri Aug 26 19:12:23 EST 1994


Hi German
I tried e-mailing you direct but my message was returned so I will have to
reply here.
I checked the NEB catalogue about Pvu II and Apa I but unfortunately they 
don't list them in their analysis of enzymes that cleave close to the ends of
linear DNA.
With regard to cleaning up your pcr product, there was a post in reply to
your original query that indicated various things in the pcr reaction may
interfere with digestion. It is probably worth trying to clean it up if you
have enough of it. I routinely purify my pcr products from agarose gels 
(not low-melting) if I have more than one product using the Qiaex kit from Qiagen (Geneclean from Bio 101 also works well if your product is >200bp). If I 
have a single product I phenol-chloroform extract then precipitate the DNA 
with 7.5M ammonium acetate. I have had no problem blunt-end ligating these 
products but I haven't tried digesting them (except for the incident with
Nde I I mentioned previously!).
If I can help any more drop me a line. Good luck
Emma



More information about the Methods mailing list