Biomedical Engineering Reference
In-Depth Information
analysis, sequencing data analysis and contig assembly, virtual
plasmid construction, direct access to NCBI databases, and is
compatible with multiple fi le-types. It is a multi-platform pro-
gram (Java-based), and the user interface is intuitively designed.
4. Ending a primer on the 3
end with one to two G/C pairs
(GC-clamp) will increase PCR yield and performance as GC
hydrogen bonding is stronger than for AT pairs.
5. A strong (
T
m
>50 °C) secondary structure, especially if residing
at the 3
′
end of the primer, will greatly reduce the performance
of PCR leading to low or no product amplifi cation, and primer
replication.
6. If the template is a plasmid, linearize the plasmid if possible by
restriction digest to reduce the number of colonies after trans-
formation harboring non-SLIC plasmids. Final template con-
centrations should be approximately 20 ng/50
′
L reaction for
plasmids and 100 ng/reaction for genomic DNA.
7. Phusion polymerase has a fl exible extension time of 15-30 s/1 kb
of DNA. As a general rule, reactions with products equal to or
less than 2 kb can be completed with a 30 s extension time. It is
advised, however, to add an additional 30 s for every 2 kb of
product. For DNA fragments greater than 4 kb, use an exten-
sion time of 20 s/1 kb. In our lab, we have replicated up to
11.7 kb, with extension times of 3 min, and regularly replicate
6 kb with extension times of 2 min.
μ
8. A SLIC primer at ~40 bp will have an average
T
m
of about
70 °C and an initial binding temperature of 55-65 °C (phase
one), because only half of the primer will match the initial tem-
plate. The protocol provided is a variant of TD-PCR in that it
will slowly drop annealing temperatures for 10 cycles to allow
for initial binding, then return to a higher annealing tempera-
ture (phase two) for 15 cycles. If the PCR is unsuccessful, try
adjusting the initial binding range (phase one) from 65-55 °C
to 60-50 °C. If additional bands are observed, i.e., nonspecifi c
amplifi cation, try increasing to 70-60 °C. The initial annealing
temperature range should encompass both primers' initial
T
m
values. Phase two annealing temperature should be adjusted
such that it is 1-2 °C below the lowest
T
m
.
9. If the gel is not set, then the wells can be damaged when the
comb is removed. Additionally, the separation of product can
be impaired to the point where the bands are smeared beyond
recognition. To ensure the gel is set in a timely manner, it is
suggested that the fl ask of molten agarose be cooled by briefl y
running under a cold-water tap (1-2 min). Alternatively, sim-
ply pour the gel at the beginning of the TD-PCR protocol, as
the TD-PCR will take a minimum of 45 min to complete,
which is ample time for the gel to set.
