Biomedical Engineering Reference
In-Depth Information
Ala residue. These overhangs were selected because they code
for small neutral amino acids and are completely incompatible,
thus excluding self-ligation of the vector or concatemer forma-
tion of the insert.
7. If genomic DNA is used as a template, delicate handling is
required and care should be taken not to compromise the
material by repetitive freeze/thawing, vortexing, or vigorous
pipetting.
8. Touchdown PCR [
24
] is recommended as it favors the pro-
duction of the desired product over products resulting from
spurious priming. In addition, as a range of annealing tempera-
tures is used the program does not require much fi ne-tuning
for different targets.
9. Purifi cation of the target PCR product from gel might be
omitted if the quality of the PCR was superb. However, regu-
larly small by-products are present and even trace amounts of
these are cloned with high effi ciency. As a result, more clones
need to be analyzed in later steps thus decreasing the through-
put. This is not a unique feature of FX cloning but applies to
all (high-throughput) cloning procedures.
10. Due to the high effi ciency of FX cloning and the use of primers
with uniform restriction sites, care should be taken to avoid
cross-contamination with PCR products run in adjacent wells.
In addition, the gel container should be cleaned and the buffer
should be replaced between runs to prevent contamination
with PCR products analyzed in previous runs.
11. Should the PCR not yield the desired product in suffi cient
amounts, consider the use of alternative buffers supplied by the
manufacturer, the addition of dimethylsulfoxide (DMSO) or
the use of a freshly purifi ed template. More detailed suggestions
can be found in the molecular cloning protocol series [
25
].
12. The choice of FX cloning vector depends on the purpose. To
allow subcloning of sequence-verifi ed ORFs, clone the insert
fi rst into a pINITIAL-derivative. Preferably the antibiotic
marker of the pINITIAL-derivative is different from the
expression vectors used later for subcloning as this provides
another selection criterium next to sacB counterselection.
Derivatives of pINITIAL holding antibiotic markers against
kanamycin, chloramphenicol, or tetracycline are available.
As most common expression vectors provide resistance against
ampicillin or kanamycin, in general pINITIAL-cat (providing
chloramphenicol resistance) is recommended. If there is no
need for subcloning, the ORFs can be cloned immediately into
an FX cloning-compatible expression vector.
13. A facile approximation for a 1:5 molar ratio of vector:insert is
calculated using: (amount vector (ng)×size insert (bp)×5)/
size vector (bp) = amount insert (ng) needed.
