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dam-/dcm- Competent E. coli > FAQ |  | dam-/dcm- Competent E. coli FAQ
See the Competent Cells FAQ also.
Q1: How should I calculate the transformation efficiency (C2925)?
Q2: What are the solutions/recipes (C2925)?
Q3: What are the strain properties (C2925)?
Q1: How should I calculate the transformation efficiency (C2925)?
A1: Transformation efficiency is defined as the number of colony forming units (cfu) which would be produced by transforming 1 µg of plasmid into a given volume of competent cells. The term is somewhat misleading in that 1 µg of plasmid is rarely actually transformed. Instead efficiency is routinely calculated by transforming 100 pg-1 ng of highly purified supercoiled plasmid under ideal conditions. If you plan to calculate efficiency to compare cells or ligations, keep in mind the many variables which affect this metric.
Transformation efficiency (TE) equation:
TE = Colonies/µg/Dilution
Colonies = the number of colonies counted on the plate
µg = the amount of DNA transformed expressed in µg
Dilution = the total dilution of the DNA before plating
TE calculation example:
Transform 2 µl (100 pg) of control pUC19 DNA into 50 µl of cells, outgrow by adding 950 µl of SOC before plating 100 µl. If you count 20 colonies on the plate, the TE is:
Colonies = 20
µg DNA = 0.0001
Dilution = 100/1000 = 0.1
TE = 20/.0001/0.1 = 2 x 106 cfu/µg
Q2: What are the solutions/recipes (C2925)?
A2: SOB:
2% Vegetable peptone (or Tryptone)
0.5% Yeast Extract
10 mM NaCl
2.5 mM KCl
10 mM MgCl2
10 mM MgSO4
SOC:
SOB + 20 mM Glucose
LB agar:
1% Tryptone
0.5% Yeast Extract
0.17 M NaCl
1.5% Agar
Q3: What are the strain properties (C2925)?
A3: The properties of this strain that contribute to its usefulness as a protein subcloning strain are described below. The genotypes underlying these properties appear in parentheses.
dam and dcm Methylation Deficient (dam13::Tn9 (CamR), dcm-6)
Most laboratory strains of E. coli contain both Dam methylase and Dcm methylase. Dam methylase transfers a methyl group to the adenine in the sequence GATC. Dcm methylase methylates the internal cytosine residues in the sequences CCAGG and CCTGG. Several restriction endonucleases will not cleave sites with these modified bases. The damdcm strain allows growth and purification of DNA free of Dam and Dcm methylation
Endonuclease I Deficient: (endA) The periplasmic space of wild type E. coli cells contains a nonspecific endonuclease. Extreme care must be taken to avoid degradation of plasmids prepared from these cells. The endA mutation deletes this endonuclease and can significantly improve the quality of plasmid preparations.
Restriction Deficient: (hsdR2) Wild type E. coli K12 strains carry the EcoK Type I restriction endonuclease which cleaves DNA with sites (AAC(N6)GTGC and GCAC(N6)GTT. While E. coli DNA is protected from degradation by a cognate methyl-transferase, foreign DNA will be cut at these sites. The hsdR2 mutation described above eliminates the endonuclease.
Partially Methyl Restriction Deficient: (mcrA, mcrB1 ) E. coli has a system of enzymes, mcrA, mcrB and mrr which will cleave DNA with methylation patterns found in higher eukaryotes, as well as some plant and bacterial strains. DNA derived from PCR fragments, cDNA or DNA previously propagated in E. coli will not be methylated at these sites and will not be cleaved. This strain has a functional Mrr endonuclease and may not be suitable for direct cloning of eukaryotic DNA.
T1 Phage Resistant: (fhuA31) T1, an extremely virulent phage requires the E. coli ferric hydroxamate uptake receptor for infectivity. Deletion of this gene confers resistance to this type of phage, but does not significantly affect the transformation or growth characteristics of the cell.
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