331
protocol listed here can be used to clone three consecutive
domains leading to a single three-domain protein, or to clone
libraries in which each “AB,” “BC,” or “CD” fragment is actually a large collection of different domains sharing the same
overhangs. In the latter case, the result is not a single threedomain protein, but rather a library of multiple different combinations of three domains arranged in the desired orders. This
order is determined by the presence of consecutive matching
overhangs (e.g., “AB” will ligate upstream of “BC”).
4. Clean the PCR reactions using the QIAquick PCR cleanup kit
as indicated by the manufacturer.
5. Determine the concentration of each amplified fragment
library by measuring absorbance at 260 nm using a Nanodrop
spectrophotometer (or equivalent instrument). This step is
key, as AarI digestion is sensitive to DNA concentration.
6. For AarI-mediated multi-insert cloning of domain shuffled
libraries, it is necessary to use an acceptor vector flanked by
AarI recognition sequences with the first and last overhangs
used (“A” and “D” in our case), as show below:
5′…PROMOTER…GGAGCAAGGCAGGTG…(~20
bp
intermediate
sequence)…CACCTGCAACATGCG…
TERMINATOR…3′
Note the flanking overhangs in bold, and the AarI recognition
sequences (in opposite directions) underlined.
7. Set up individual AarI digestion reactions for each PCRgenerated DNA fragment library, as well as for the acceptor
plasmid DNA. For each reaction, set up the components as
depicted in Table 6, mix gently and incubate at 37 °C for 3 h:
8. Inactivate AarI by incubation at 65 °C for 20 min.
9. Purify digested DNA (both plasmid and PCR fragments)
directly with QIAquick PCR clean up kit. Note that as long as
the fragment released from the acceptor vector is small (up to
20 bp) there is no need for gel purification.
Table 6
Setup for AarI restriction enzyme reaction
Component
Amount
DNA (either acceptor plasmid OR PCR fragment)
5 μg
10× AarI reaction buffer
6 μL
50× AarI oligonucleotide (0.025 mM)
1 μL
AarI enzyme
2.5 μL
Water
To complete 60 μL
Rewiring Signaling Networks
protocol listed here can be used to clone three consecutive
domains leading to a single three-domain protein, or to clone
libraries in which each “AB,” “BC,” or “CD” fragment is actually a large collection of different domains sharing the same
overhangs. In the latter case, the result is not a single threedomain protein, but rather a library of multiple different combinations of three domains arranged in the desired orders. This
order is determined by the presence of consecutive matching
overhangs (e.g., “AB” will ligate upstream of “BC”).
4. Clean the PCR reactions using the QIAquick PCR cleanup kit
as indicated by the manufacturer.
5. Determine the concentration of each amplified fragment
library by measuring absorbance at 260 nm using a Nanodrop
spectrophotometer (or equivalent instrument). This step is
key, as AarI digestion is sensitive to DNA concentration.
6. For AarI-mediated multi-insert cloning of domain shuffled
libraries, it is necessary to use an acceptor vector flanked by
AarI recognition sequences with the first and last overhangs
used (“A” and “D” in our case), as show below:
5′…PROMOTER…GGAGCAAGGCAGGTG…(~20
bp
intermediate
sequence)…CACCTGCAACATGCG…
TERMINATOR…3′
Note the flanking overhangs in bold, and the AarI recognition
sequences (in opposite directions) underlined.
7. Set up individual AarI digestion reactions for each PCRgenerated DNA fragment library, as well as for the acceptor
plasmid DNA. For each reaction, set up the components as
depicted in Table 6, mix gently and incubate at 37 °C for 3 h:
8. Inactivate AarI by incubation at 65 °C for 20 min.
9. Purify digested DNA (both plasmid and PCR fragments)
directly with QIAquick PCR clean up kit. Note that as long as
the fragment released from the acceptor vector is small (up to
20 bp) there is no need for gel purification.
Table 6
Setup for AarI restriction enzyme reaction
Component
Amount
DNA (either acceptor plasmid OR PCR fragment)
5 μg
10× AarI reaction buffer
6 μL
50× AarI oligonucleotide (0.025 mM)
1 μL
AarI enzyme
2.5 μL
Water
To complete 60 μL
Rewiring Signaling Networks