Biomedical Engineering Reference
In-Depth Information
Reaction Nr.
1
2
STOP dNTP
dTTP
dATP
DNA fragments
composite fragment 1
composite fragment 3
composite fragment 2
BB 2
2. Transform the reaction mix into chemo-competent
E. coli
and
inoculate liquid cultures as described.
Note 4
provides additional information for the design of the
second-level backbone in order to maximize the fi delity of the
cloning process.
3. Prepare the plasmid DNA using a miniprep kit.
3.9
Quality Control
1. Check the second-level assembly clones for being correct by
mixing 5
μ
L plasmid DNA solution with 1
μ
L 10× FastDigest
green buffer, 3
L FastDigest restriction
enzyme of choice. Incubate for 30 min at 37 °C and analyze
on a 1 % agarose gel for the correct band pattern as predicted
using the software “Ape Plasmid Editor”.
μ
L H
2
O, and 2× 0.5
μ
4
Notes
1. Due to the requirement for an alternating use of the extension
nucleotides, only even numbers of DNA fragments can be assem-
bled in a single tube. This requirement can be omitted by intro-
ducing short paired oligonucleotide fragments bearing
appropriate overhangs. Two oligonucleotides should be designed
such that their 3
′
ends are reverse complementary for at least 20
bases and their 5
ends are complementary to the overhangs gen-
erated by T4 DNA polymerase chew-back of PCR products. The
oligonucleotides should be added at a similar molarity to the
PCR fragments after the chew-back has been heat-inactivated,
since otherwise they could be subject to rapid degradation.
′
2. By digesting the PCR products with DpnI prior to the assembly,
the cloning background due to template plasmid DNA contam-
ination bearing the same resistance gene as the fi nal assembly
construct can be reduced. DpnI digests only methylated plasmid
DNA of bacterial origin, while PCR products are left intact.
3. The type-IIs restriction enzyme AarI can be replaced by other
enzymes. If no outside cutter is used, the sequence portion of
the enzyme's recognition site which is retained after cutting
and 3
chew-back determines parts of the ID sequences to be
used. Importantly, restriction enzymes leaving 3
′
overhangs
generally predetermine less of the ID sequences than enzymes
leaving 5
′
overhangs are removed at the
time of chew-back irrespective of their base composition.
Suitable alternatives to AarI are PacI, BsmBI, and BsmI.
′
overhangs, since 3
′
