I have tried all sorts of concentration variables of SAM, Mme1, and the target DNA. It works well on the pUC19 plasmid which has two Mme1 sites but gives 0% activity on a 100 bp PCR fragment having one and two Mme1 sites. Any suggestions?
Thanks Michael,
Actually I am ligating a 30 bp linker containing Mme1 site to a 100bp DNA fragment. Linker is made in such a way that only one side of it will ligate and this side has Mme1 site at the extreme end. Ligation is working nicely, but now the Mme1 is not cutting at all. On the contrary, I am using a plasmid containing Mme1 site as a positive control. Mme1 is cutting the plasmid nicely, but not the ligated 130bp product. As I mentioned, after ligating, the Mme1 will have 100 bp span to cut so length is not a problem unless the Mme1 recognition and binding to DNA is not directional.
The linker is not a PCR amplified product, it is made simply by annealing two oligos which were gel purified after synthesis and then annealed.
Also, I am purifyng the DNA after ligation using PCI and ethanol precipitation.
Some restriction enzymes need two sites to be present in the same DNA molecule to cut at all. SfiI is like this. Plasmids with ine site are not cut, those that have two sites are cut at both site efficiently.
Hi, Sanjay, has you solved this problem? I am working on Chia-pet, which has the similar process like yours, each half linker contains one mme1 recognition sites, so one full linker will have 2 sites separated by 18bps. I am not sure if the mme1 cutting part in my experiment worked or didn't, but the final result is this assay failed.
mme1 will self block if excessive enzyme is used, it will methylate the dna other than cutting. in my protocol, half linkers were added to quench the excessive enzyme in the cutting mixture, so it is like hijacking. But I have no idea, in such condition, how many functional molecules of enzyme still do the job. By calculation, excessive amount could be avoided, but I just followed the protocol last time, I haven't modified anything yet.
Hi Yu,
No, I was not able to solve the problem. Additionally, my linkers were self ligating. Due to the small size of linker-dimer, it was getting cut with Mme1 more efficiently compared to my target genomic DNA. This lead to cloning and sequencing of majorly the linker itself instead of the target DNA. Then there were other issues with the overall purpose of my experiment and ultimately I had to re-design the whole strategy. I am not using Mme1 anymore. Sorry.
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