It seems that XbaI is blocked by overlapping dam methylation in this case. If the recognition site is preceded by GA or followed by TC, dam methylase GATC will overlap the XbaI site TˆCTAGA: blocked area ga TˆCTAGA and TˆCTAGAtc. There are two alternative options: (1) Use a dam-deficient strain like GM2163((E4105S) or JM110 cell to amplify the DNA firstly before digestion, (2)Amplify the insert by PCR and digest it by XbaI to avoid the XbaI methylation.
Articles in this section
- Why can't I cut this plasmid with XbaI and I can successfully cut other constructs with XbaI?
- Why customers get incorrect colonies when the CloneEZ reaction is transformed?
- Why customers are not able to get sufficient colonies when the CloneEZ reaction is transformed?
- Why do you add some extra bases outside of cloning sites? Do these bases interfere with any downstream applications?
- What's the method Genscript uses for subcloning?
- What is your definition of “subcloning” , “PCR cloning” and “customized cloning”?
- What is your cloning/subcloning method in generating constructs for customer?
- What is the main difference between cloneEZ and Invitrogen Gateway cloning?
- I need a common expression vector. Do you have any in stock?
- What E.coli competent do you usually use for cloning?