Troubleshooting guide

40 QIAprep Miniprep Handbook 12/2006
Host strains
Most
E. coli strains can be used successfully to isolate plasmid DNA, although the strain
used to propagate a plasmid has an effect on the quality of the purified DNA. Host
strains such as DH1, DH5α, and C600 give high-quality DNA. The slower growing
strain XL1-Blue also yields DNA of very high-quality which works extremely well for
sequencing. Strain HB101 and its derivatives, such as TG1 and the JM series, produce
large amounts of carbohydrates, which are released during lysis and can inhibit enzyme
activities if not completely removed (4). In addition, these strains have high levels of
endonuclease activity which can reduce DNA quality. The methylation and growth
characteristics of the strain should also be taken into account when selecting a host strain.
XL1-Blue and DH5α are highly recommended for reproducible and reliable results.
Inoculation
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 may lead to uneven plasmid yield or 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 so 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 1–5 ml of
media containing the appropriate selective agent, and grown with vigorous shaking for
12–16 hours. Growth for more than 16 hours is not recommended since cells begin to
lyse and plasmid yields may be reduced.
Antibiotics
Antibiotic selection should be applied at all stages of growth. Many plasmids in use
today do not contain the
par locus which ensures that the plasmids segregate equally
during cell division. Daughter cells that do not receive plasmids will replicate much
faster than plasmid-containing cells in the absence of selective pressure, and can quickly
take over the culture.
The stability of the selective agent should also be taken into account. Resistance to
ampicillin, for example, is mediated by β-lactamase which is encoded by the plasmid-
linked
bla gene and which hydrolyzes ampicillin. Levels of ampicillin in the culture
medium are thus continually depleted. This phenomenon is clearly demonstrated on
ampicillin plates, where “satellite colonies” appear as the ampicillin is hydrolyzed in
the vicinity of a growing colony. Ampicillin is also very sensitive to temperature, and
when in solution should be stored frozen in single-use aliquots. The recommendations
given in Table 6 are based on these considerations.