Hi all!
I tried to prepaare a library for genome wide methylation analysis with RRBS.
Unfortunatly I saw that MspI is a very very annoing enzyme which digest very slowly and unefficiently. The Meissner paper which reported this tecnique said that they use 5-20 U of enzyme for 30 ng-1 micrograms (nat methods 2010) or 10-100 U of enzyme for 1-10 micrograms (nature 2008) of genomic DNA. Here in institute, to obtain a "smear" of digested DNA (visible only upper 700 bp) we use 100-300 U for 1 microgram of DNA and digested overnight. We tried a lot of things ad the best seems this, far from conditions disclaimend in the paper..Is there someone who can suggest me somenthing new?
I load into gel 1 micrograms of digested dna and purified the "gel zone" (when i never had seen any smear) 100-400 bp and I obtained only 1 ng of DNA!!!!!
And if I would digest twice the dna (that is digest and re-digest?)
please help me
I tried to prepaare a library for genome wide methylation analysis with RRBS.
Unfortunatly I saw that MspI is a very very annoing enzyme which digest very slowly and unefficiently. The Meissner paper which reported this tecnique said that they use 5-20 U of enzyme for 30 ng-1 micrograms (nat methods 2010) or 10-100 U of enzyme for 1-10 micrograms (nature 2008) of genomic DNA. Here in institute, to obtain a "smear" of digested DNA (visible only upper 700 bp) we use 100-300 U for 1 microgram of DNA and digested overnight. We tried a lot of things ad the best seems this, far from conditions disclaimend in the paper..Is there someone who can suggest me somenthing new?
I load into gel 1 micrograms of digested dna and purified the "gel zone" (when i never had seen any smear) 100-400 bp and I obtained only 1 ng of DNA!!!!!
And if I would digest twice the dna (that is digest and re-digest?)
please help me
Comment