demethylases - (Jul/19/2005 )
I'm just wondering whether it wouldn't be better to use genomic DNA from 5-aza-cytidin-treated cells as a negative control when someone is studying gene methylation?
Now I have more time to explain. I should first apologize because last time I did not explain correctly.
First, You can get free dam- dmc- cells from Fermentas
E.coli GM2163 dam-, dcm- is available for free upon request, Catalog number #M0099
http://www.fermentas.com/techinfo/re/restrdamdcmmeth.htm
Regarding cloning of methylated DNA
Of the following E.coli cells (invitrogen catalog number):
One Shot ® MAX Efficiency ® DH5 ™ T1 (12297-016)
MAX Efficiency ® DH5 ™ T1 Phage-Resistant a (12034-013)
MAX Efficiency ® DH5 ™ (18258-012)
Library Efficiency ® DH5 ™ (18263-012)
Subcloning Efficiency ™ DH5 ™ (18265-017)
MAX Efficiency ® DH5 F´IQ ™ (18288-019)
UltraMAX ™ DH5 -FT ™ (10643-013)
ElectroMAX ™ DH5 -E ™ (11 319-019)
just ElectroMAX ™ DH5 -E ™ (11 319-019) does NOT restrict methylated DNA, therefore if you are going to clone methylated genomic DNA or cDNA this is the only strain in the DH5 alpha group that results in better represented libraries
Regarding production of unmethylated DNA
all of the DH5 alpha strains from invitrogen produce dam/dmc methylated DNA (althoug it is not CpG phosphorylated). If you need to produce dam/dmc unmethylated DNA (if you are going to use a dam/dmc sensitive restriction enzyme) you should use INV 110 or the dam negative dmc negative E. colli strain from fermentas.
the sequencing of DNA produced with any of the DH5 strains in a region that does not contain dam/dmc methylated sequences does not represent any problem, the problem could arise if the sequence to be sequenced contains a dam/dmc methylation