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?
- 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?
- What is the antibiotic resistance of the vector pUC57?
- There is a restriction site in my gene, but I want to clone the gene into a vector using this site. Can you clone it for me?
- On the order form, it requires cloning sites. Is this which cloning sites I would like the gene inserted into the vector? Also, do I have to include these sites at the both ends of my sequence when ordering?