It has been found that storing dam strains on agar medium (i.e., on plates or in slope or stab culture) for periods longer than approximately one month results in strain instability.
The most suitable method of storage for these strains is to keep aliqouts of cell suspensions in broth containing 40% glycerol at -70 C, under which conditions they are stable and viable for many years.
Testing for dam
The presence of the mutant dam allele in a strain can be verified in a number of ways.
1. Isolate chromosomal and/or plasmid DNA and test susceptibility to digestion with DpnI (sensitive) and DpnII (resistant).
2. Test for mutator phenotype. Rifampicin-resistance as a marker is an easy, reliable and foolproof test.
3. Test for sensitivity to 2-aminopurine.
dam alleles (MG1655 U00096.2)
dam-3 - a GC to AT transition mutation at nt 3,513,898 changing GGC (gly13) to GAC (aspartate)
dam-4 - a GC to AT transition mutation at nt 3,513,901 changing GGG (gly12) to GAG (glu)
dam-12::Mud (Ampicillin resistance)
dam-13::Tn9 (Chloramphenicol resistance) - an insertion between nt 3,513,725 and 3,513,726
dam-16::Kan (Kanamycin resistance) - kanamycin-resistance gene inserted between and replacing nt 3,513,240 and 3,513,773
Working with dcm strains
Strains with dcm mutations are quite stable and need no special handling or storage conditions. Colonies on plates at 4 C can be used for several months. We routinely keep aliqouts of cell suspensions in broth containing 40% glycerol at -70 C.
Testing for dcm
1. Isolate plasmid and/or chromosomal DNA and test susceptibility to digestion with EcoRII (or ScrFI) and BstNI.
2. Prepare lysates of phage lambda vir on candidate strains and test efficiency of plating on strain GM1212. Phage grown on dcm mutants have a low efficiency of plating when tested on GM1212.
dcm alleles
The most widely used allele is dcm-6. It results in complete loss of detectable 5-methylcytosine in DNA. It also inactivates the vsr gene.