The table below gives the concentrations of commonly used antibiotics. Ampicillin is also very sensitive to temperature, and should be stored frozen in single-use aliquots. It is important to inoculate cultures from freshly prepared plates to ensure that the antibiotic is effective. This phenomenon is clearly demonstrated on ampicillin plates, where "satellite colonies" appear as the ampicillin is hydrolyzed in the vicinity of a growing colony. Levels of ampicillin in the culture medium are thus continually depleted. Resistance to ampicillin, for example, is mediated by β-lactamase which is encoded by the plasmid-linked bla gene and which hydrolyzes ampicillin. The stability of the selective agent should also be taken into account. Daughter cells that do not receive plasmids will replicate much faster than plasmid-containing cells and can quickly take over the culture. Many plasmids in use today do not contain the par locus which ensures that the plasmids segregate equally during cell division in the absence of selective pressure. It is often convenient to grow the starter culture during the day and the larger culture overnight for harvesting the following morning.Īntibiotic selection should be applied at all stages of growth. 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). 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). This procedure should then be repeated to ensure that a single colony of an antibiotic resistant clone can be picked.
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. Inoculation from plates that have been stored for a long time may also lead to loss or mutation of the plasmid. Subculturing directly from glycerol stocks, agar stabs, and liquid cultures is poor microbiological practice and may lead to loss of the plasmid.
If difficulty is encountered with strains such as TG1 and Top10F', we recommend either reducing the amount of culture volume used for cleared lysate preparation, or using the same amount of culture volume but doubling the volumes of Buffers P1, P2, and P3 to improve the ratio of biomass to lysis buffers for optimized lysis conditions.īacterial cultures for plasmid preparation should always be grown from a single colony picked from a freshly streaked selective plate. If after performing a QIAGEN plasmid preparation, the quality of purified DNA is not as expected, a change of host strain should be considered. The methylation and growth characteristics of the host strain can also affect plasmid isolation. In addition, some strains such as JM101, JM110, and HB101 have high levels of endonuclease activity, and yield DNA of lower quality than that prepared from strains such as XL1-Blue, DH1, DH5α, and C600. Strain HB101 and its derivatives, such as TG1 and the JM100 series, contain large amounts of carbohydrates that are released during lysis and can inhibit enzyme activities if not completely removed. The slower growing strain, XL1-Blue, also yields DNA of very high quality which works extremely well for sequencing. Host strains such as DH1, DH5α, and C600 yield high-quality DNA with QIAGEN protocols. coli strains can be used successfully to isolate plasmid DNA, although the strain used to propagate a plasmid can have a substantial influence on the quality of the purified DNA. For purification of P1 and BAC DNA using QIAGEN-tips, please contact our technical service departments or your local distributor. Cosmid DNA prepared with QIAGEN-tips is suitable for all applications including sequencing (manual or automated). See the detailed protocol in the QIAGEN Plasmid Purification Handbook. A few changes in the procedure are necessary to obtain optimal results. To obtain good yields of very low-copy cosmids, it is often necessary to use culture volumes much larger than those normally recommended for use on QIAGEN-tips. Cosmids should be treated as low- or very low-copy plasmids when determining which QIAGEN-tip to use.
Like plasmids, cosmids vary in copy number, depending on the origin of replication they contain, their size, and the size of insert. Due to their relatively large size and slow replication time, cosmids are generally present in low or very low copy numbers in bacterial cells (see table Origins of replication and copy numbers of various plasmids and cosmids). QIAGEN Plasmid Kits are also highly suited for purification of cosmid DNA.
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