Plasmid copy number
Note: The copy number of plasmids and cosmids can be substantially influenced by the cloned insert. For example, a high-copy pUC plasmid may behave like a medium- or low-copy plasmid when containing certain inserts (e.g., very large DNA fragments), resulting in lower DNA yields than expected.
Origins of replication and copy numbers of various plasmids and cosmids
|Origin of replication||Copy number||Classification|
|pBluescript vectors||ColE1||300–500||High copy|
|pGEM vectors||pMB1*||300–400||High copy|
|pTZ vectors||pMB1*||>1000||High copy|
|pBR322 and derivatives||pMB1*||15–20||Low copy|
|pACYC and derivatives||p15A||10–12||Low copy|
|pSC101 and derivatives||pSC101||~5||Very low copy|
Cosmid copy number
Bacterial cultures for plasmid preparation should always be grown from a single colony picked from a freshly streaked selective plate. Subculturing directly from glycerol stocks, agar stabs, and liquid cultures is poor microbiological practice and may lead to loss of the plasmid. Inoculation from plates that have been stored for a long time may also lead to loss or mutation of the plasmid. The desired clone should be streaked from a glycerol stock onto a freshly prepared agar plate containing the appropriate selective agent such that single colonies can be isolated. This procedure should then be repeated to ensure that a single colony of an antibiotic resistant clone can be picked. A single colony should be inoculated into 2–10 ml of LB medium (see table Composition of Luria Bertani medium) containing the appropriate selective agent and grown for ~8 hours (logarithmic phase). Using a vessel with a volume of at least four times greater than the volume of medium, the starter culture should then be diluted 1/500 to 1/1000 into a larger volume of selective medium, and grown with vigorous shaking (~300 rpm) to saturation (12–16 hours). It is often convenient to grow the starter culture during the day and the larger culture overnight for harvesting the following morning.
Concentrations of commonly used antibiotics
||Concentration of stock solution||Storage||Working concentration
|50 mg/ml in water||–20°C||100 µg/ml (1/500)|
|Chloramphenicol||34 mg/ml in ethanol||–20°C||170 µg/ml (1/200)|
|Kanamycin||10 mg/ml in water||–20°C||50 µg/ml (1/200)|
|Streptomycin||10 mg/ml in water||–20°C||50 µg/ml (1/200)|
|Tetracycline HCl||5 mg/ml in ethanol||–20°C||50 µg/ml (1/100)|
QIAGEN protocols are optimized for use with cultures grown in standard Luria Bertani (LB) medium (see table Composition of Luria Bertani medium), grown to a cell density of approximately 3–4 x 109 cells per ml. We advise harvesting cultures after approximately 12–16 hours of growth, which typically is the transition from logarithmic into stationary growth phase (see figure Growth curve of E. coli in LB medium). At this time, the ratio of plasmid DNA to RNA is higher than during the logarithmic phase. Also, the DNA is not yet degraded due to overaging of the culture, as in the later stationary phase. Please note the maximum recommended culture volumes given at the beginning of each protocol.
Several of the current bacteria strains can grow to very high cell densities. It is best to assess the cell density of the culture and reduce the culture volumes accordingly or increase the volumes of lysis buffers P1, P2 and P3, if necessary. A high ratio of biomass to lysis buffers will result in poor lysis conditions and subsequently low DNA yield and purity. If working with low-copy vectors, it may be beneficial to increase the lysis buffer volumes to increase the efficiency of alkaline lysis, and thereby the DNA yield. In case additional Buffers P1, P2, and P3 are needed, their compositions are provided in the handbooks. Alternatively, the buffers may be purchased separately.
It is not recommended to use super rich growth media such as TB (terrific broth) or 2x YT for most commonly used high-copy plasmids. Although TB or 2x YT have the obvious advantage of producing more bacteria (2–5 times), this does not necessarily lead to greater yields or higher-quality DNA.
If rich media must be used, the culture volume should be reduced to match the recommended cell biomass, which in turn should correspond to the capacity of the QIAGEN-tip used. If the culture volume used is too high, alkaline lysis will be inefficient, resulting in lower yield than expected. Furthermore, the excessive viscosity of the lysate will require vigorous mixing, resulting in shearing of bacterial genomic DNA and subsequent contamination of the plasmid DNA.
Composition of Luria Bertani medium
|Yeast extract||5 g