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
- What information do you need to prepare a quote?
- If you cannot finish the project (molecular biology project), do I still have to pay?
- I only have the vector map but not the sequence. Can you still process my request?
- Do you sequence verify the clones you made?
- Why I can't cut this plasmid (with XbaI siteTCTAGA) with XbaI while I can cut other constructs with Xba I fine?
- Why is my gene sequence different from the standard sequence?
- We have verified the insertions in the samples before submitting orders. Why the sequencing result indicates an empty vector?
- If client does not know the restriction enzyme sites flanking the genes, nor the vector map, but could provide the gene sequence, and primer sequences to be used for sequencing confirmation, can we take the project?
- With what vectors do we ship bacstab & induction solution to the client?
- Why did my PCR reaction stop working?