Biomedical Engineering Reference
In-Depth Information
2. The appropriate carbon source depends on the strains and its
ability to grow on non-fermentable conditions. When working
with strains that are unable to respire, the use of galactose is
preferred to glucose, as glucose-repression of many mitochon-
drial genes leads to poorly developed mitochondria and conse-
quently a rather poor labeling. When working with strains that
are able to respire lactic acid, glucose, galactose, and lactic acid
are added to a final concentration of 2 % (w/v). Raffinose can
also be used at a final concentration of 1 % (w/v).
3. Auxotrophic markers (amino acids or nucleotides) might be
omitted to select for plasmids.
l
l-tryptophan turns into a
brown solution during the autoclaving process; alternatively it
can be sterilized by iltration through a 0.22 ʼm ilter.
l
-tryptophan and
l
l-histidine-HCl are photosensitive and there-
fore the stock solutions must be kept protected from light.
4. PMSF is dissolved at 200 mM in ethanol. Prepare freshly
before use. PMSF inhibits serine proteases irreversibly and is
toxic. Appropriate safety measurements must be taken when
handling it.
5. We usually clone open reading frames (ORFs) into pGEM
vectors (Promega) downstream of the SP6 RNA polymerase
transcription initiation site. This generally produces transcripts
more efficiently than T3 or T7 RNA polymerases. However,
the latter enzymes were successfully used as well. It is advisable
to inspect the sequence of the mature protein for the presence
of methionine residues. To improve the labeling efficiency
additional ATG codons might have to be inserted into the
DNA sequence or at the 3′-end of the gene during the cloning
procedure.
6. Spermidine is made from a 200 mM stock solution. Spermidine
may deaminate. Thus, make a small stock solution, keep it at
−20 °C, and prepare new solutions frequently.
7. m
7
G(5′)ppp(5′)G is an mRNA cap analog, which is used
during the in vitro transcription reactions in order to yield
RNA capped at the 5′-end. This cap is required for satisfactory
in vitro translations. m
7
G(5′)ppp(5′)G is shipped in packages
with 25
A
250
units.
8. Avoid multiple freezing and thawing of the lysate. This strongly
reduces the translation efficiency. We usually do not freeze the
same aliquot more than twice. Lysate is thawed very rapidly by
hand warming and subsequently placed on ice.
9. The use of radiolabeled cysteine is possible as well. If cysteine
is used, the chase is performed with cold cysteine instead of
methionine. We also use radiolabeled methionine-cysteine
mixes. This can enhance the labeling efficiency.
