Biomedical Engineering Reference
In-Depth Information
suffi cient to obtain ample but distinct colonies for subsequent
analysis. However, if no upper DNA band is observed (
see
Subheading
3.2
,
Note 7
, and Fig.
3a
, lane 1), it is recom-
mended to use the entire 10
9. Colony PCR is an optional step, although highly recom-
mended. Before selecting clones for DNA sequencing it is
advisable to perform colony PCR to ensure that the selected
clones harbor the target gene. The colony PCR reactions are
carried out using forward and reverse primers, derived from
sequences fl anking the cloning sites in the vector (
see
Table
1
).
Alternatively, a combination of a specifi c primer from the insert
and a primer from the expression vector is used. It is advisable
to perform a negative control PCR reaction where the destina-
tion vector without the target gene is used instead of the clonal
DNA. Before performing the PCR reaction, it is essential to
maintain the selected clones by striking on a selective plate or
into a liquid media.
10. The absence of colonies following transformation step is a clear
indication for a failure either at the cloning stage or in the
transformation procedure. In order to identify the source of
failure follow the following guidelines: Remove 1-2
μ
L reaction for transformation.
L from
the TPCR reaction and perform PCR reaction and analysis as
described in Subheading
3.3
, using fl anking primers (T7 and
PetRev, in the case of cloning into pET-derived vectors).
Analyze PCR reaction on agarose gel.
μ
If a PCR product at the expected size is observed, the TPCR
reaction was successful. In this case, the transformation stage
has failed. Reexamine the effi ciency of the competent cells
(
see
Note 3
) and make sure that the antibiotic used for selec-
tion was correct. An additional possibility for failure is that
the gene product cloned is toxic to the bacterial cells and its
expression, even at very low levels, is detrimental to cell
growth. In this case, addition of 1 % glucose to the selection
plates may alleviate the problem. However, re-cloning of
your gene into a tighter expression system may be required
to obtain transformants following the cloning stage.
●
If no PCR product was obtained it is likely the TPCR reac-
tion has failed. In this case reexamine the primers designed
for the TPCR cloning (
see
Subheading
3.1
,
Note 2
, and
Fig.
2
). To determine if the fi rst stage of the TPCR reac-
tion (megaprimer synthesis) worked, set up PCR reaction
as described in Subheading
3.2
but without the recipient
plasmid. Analyze 2-3
●
L from the reaction on 1 % agarose
gel. If following the agarose gel analysis no band at the
expected size of the target gene is observed it is likely that
the reaction conditions should be changed (e.g., add
DMSO 5-10 % (
v
/
v
)). However, if a PCR product corre-
sponding to the size of the target gene is obtained without
μ
