I recently found a phenomenon that I was working on genetic engineering. the plasmid vector was cut with notI and xbaI, and the insert segment PCR is also flanked by these two sites. but there is a TGA right in front of 3' xbaI. so the insert is in this format:
notI---GOI---TGAtctaga (xbaI:tctaga)
genetic engineering was successful and positive colonies confirmed. However after this, I found the xbaI site is no longer cutable. even weird is that, my sequence reading software stops detecting the xbaI site, even tho- the xbaI sequence is right there. (and you can try put TGATCTAGA into your own sequence software it probably wont recognize xbaI)
I wonder if the TGA somehow disrupted the xbaI cutting ability, or any other property I havent heard about xbaI that causes this?
XbaI is blocked by overlapping methylation, directed by the GATC sequence (methylation of the adenine moiety), in E. coli. If you propagate your plasmid in a methylation-deficient host such as JM110, you're plasmid will cut normally with XbaI. In methylation-competent strains, no digestion will be possible.
I have a question, is the TGA was in your PCR fragment before cloning, for example as the end of your DNA fragment?
if you cut your PCR fragment with xbal, so xbal had worked before on the same sequence before; because the PCR fragment before cutting it with xbal it should had this seq. TGA+the xbal recognition site (tctaga) so after digestion the results seq. should be TGA(same from your PCR fra.) + t (from the xbal rec. site) ...... TGAt . Then you ligated with the plasmid which was digested also with xbal which means that the sticky end should had this seq. ctaga, and the ligation would happen between -TGAt (from PCR fra.) and ctaga (from plasmid), please correct me if I misunderstood you.
if that what happened, you should not have any problems with digesting this seq again with xbal . but if after the cloning was successful, xbal is not recognizable in your sequence, this should had another explanation. for example did you check for point mutation in xbal site after cloning ?
That's an excellent question. The PCR fragment should come with TGA-xba sequence as well. However after ligation, the inserted piece was confirmed by PCR and digestion (with different enzymes other than xbaI), thus it seems at the PCR stage xbaI cuts fine. However, the TGA-xbaI containing plasmid loses the xbaI site.
I've been playing with my sequence analyze software. that TGAtctaga will result an additional digestion site as DpnI (GA^TC). adding any base between TGA and tctaga will re-activate xbaI site. However I still dont understand how overlapping DpnI will disrupt xbaI to be cut.
XbaI is blocked by overlapping methylation, directed by the GATC sequence (methylation of the adenine moiety), in E. coli. If you propagate your plasmid in a methylation-deficient host such as JM110, you're plasmid will cut normally with XbaI. In methylation-competent strains, no digestion will be possible.
You can use free online tools at http://www.molbiotools.com
In the DNA sequence editor (WebDSV) you will see all XbaI sites and those blocked by overlapping dam methylation will be marked by asterisk.
Daniel has said it all. Your problem is just the dam methylation which blocked the XbaI cleavage activity. Typically, whenever Xba I site is preceeded by GA or followed immediately by TC, this problem occurs. I experienced that problem recently while doing double digestion of some plasmids constructs with XbaI and XhoI. I was suspecting ligation inefficiency but later confirmed that it was due to this methylation. If you must use the startegy with GATC, its better to use other strains of E.coli such as JM110 or GM2163 that are dam deficient as your cloning host, but not top10.
Best wishes
To learn about the correct strains for different types of plasmids check out this Plasmids 101 blog http://blog.addgene.org/plasmids-101-common-lab-e-coli-strains
(or download the free Plasmids 101 ebook - if you are going to use plasmids, may as well know a lot about how they work!)
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