ligation problem - (Jun/18/2002 )

i construct a vetor ,(insert 300bp,vector 6.4bp,kna resistence) insert is pcr amplified with bamhi &noti rz site and 3 additional bp to the ends,then ethnol purify it  i think it can be degested perfectly and so with vector . and 5.8bp band is recovered with qiaxII kit.    i  think it is no problem ,then ligation in the rt for 3hours.  
in -20OC keeping.then DnD method for transmant Ecoli cell(DH5@) it is ok too.in kan(20ug/ml)lb plate for 16~longer but that is the problem  no bac dot on it .but strangely a few days latter , dots appears.but on kan resistence.what is wrong ? and pcr detect 300bp dot ,the contrast appears 300bp too,imaginable !!!
what contamination ,the remaining of ligation 300bp pcr product? any one can help me / please .urgent!!!
i am going crazy!

Hi,

I do not really understand all the content of your message, but you tried to digest a PCR product (300 bp) containing a NotI site with 3 flanking nucleotides. Well, I think this is not possible, this enzymze needs at least 8 flanking bases to reach 90% digestion on 20 hours....(have a look to ie: New England Biolabs catalog, 2000-01, page 210).

You can detect 300 bp by pcr maybe because the product which has been only single digested and ligated with the vector leads to a linear DNA molecule which has very low transforming properties, but has some...
These bacteria does not resist to Kan, this is the reason why they appear after Kan degrades...(a few days @ 37°C).

So don't be crazy and do not use NotI in these conditions, try to blunt it or add linkers DNA...

Good Luck and have fun !!

to:sangno
thank you!
i know your meaning!but it seems a nucleatideabout 22bp length ,the message following it is the condition of dna eds about re digestion ,it means that can be digested with noti with 3 flanking nuceatides!
what is the difference?