Working
Escherichia coli K-12 dam, dcm and mut strains
Escherichia coli K-12 dam, dcm and mut strains
- Working with dam strains
- Storage
- 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.