DH11S: an Escherichia coli strain for preparation of single-stranded DNA from phagemid vectors.
A new E. coli strain DH11S [mcrA delta(mrr-hsdRMS-mcrBC) delta(lac-proAB) delta(rec1398) deoR rpsL srl- thi-/F'proAB+ lacIqZ delta M 15] has been constructed. Transformation of DH11S competent cells with any of several different phagemid vectors [pSPORT1, pBluescript II SK(+), pGEM11Zf(+)] results in the production of highly purified single-stranded DNAs upon the addition of M13KO7 helper phage. Contamination by double-stranded DNAs was observed with all the other studied strains (XL1-Blue, JM109, DH5 alpha F'IQ). The optimal yield of single-stranded DNA production was obtained when glycerol stocks made from stationary phase cells or single colonies from overnight ampicillin plates of DH11S containing the phagemid vector were infected with M13KO7 helper phage using a wide range (1 to 100) of multiplicities of infection. Five different pSPORT1 clones containing cDNA inserts of various lengths (0.3 kb to 2.0 kb) were compared using these four different bacterial strains. The use of strain DH11S results in the best yields and quality of single-stranded DNA. Therefore, DH11S appears to be the best all-around host for various applications that require single-stranded DNA such as DNA sequencing, in vitro mutagenesis and construction of subtractive cDNA libraries.
Lin
J J
JJ
Molecular Biology Research and Development, Life Technologies, Inc., Gaithersburg, MD 20877.
Smith
M
M
Jessee
J
J
Bloom
F
F
D003090:Coliphages / Q000235:genetics; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004277:DNA, Single-Stranded / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005822:Genetic Vectors / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D000091243:Ribosomal Protein S9; D014169:Transformation, Bacterial
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D000091243:Ribosomal Protein S9; C000717190:RpsI protein, E coli