3.4 SLIC Reaction
As mentioned above, different approaches can be used to assemble
DNA fragments into a linearized vector containing overlapping
ends. Sequence and ligation–independent cloning (SLIC), in contrast to most other homology-based cloning strategies, does not
necessitate specific kits and only requires T4 DNA polymerase to be
tested first.
1. Set up the T4 DNA polymerase treatment for the four PCR
products in separate tubes. In each tube, mix 1 μg of DNA,
3 μL of 10Â NEB 2.1 buffer, and 0.5 U of T4 DNA Polymerase in a final volume of 30 μL. Incubate the reaction for 30 min
at room temperature (see Note 4).
2. Stop the exonuclease reaction by adding 3 μL of a 10 mM
dCTP solution (1/10 of the reaction volume) and leave on
ice. In the absence of dNTPs, the T4 DNA polymerase has a 3
0
to 5
0 exonuclease activity and “chews” the DNA leaving a
single-stranded region. Addition of dCTP stops the exonuclease activity and shifts it to a polymerase activity at the first
occurrence of this nucleotide.
3. Prepare an annealing mix containing the polymerase-treated
vector backbone, the dual promoter unit, and the two cDNAs
in a 1:2:2:2 ratio using 250 ng (0.15 pmoles) of a 3 kb vector
and appropriate amount of inserts (see Note 5), in a total
volume of 20 μL. Incubate the annealing mix at 37
C for
30 min and leave on ice until transformation or store at
À20
C. A negative control where one of the insert is omitted
can be included to evaluate background.
4. Use 10 μL of the annealing reaction to transform 100 μL of
E. coli competent cells (do not forget to use Pir1 cells for
plasmids with an R6Kγ replication origin). Plate the transformation mixture on LB agar plates containing the appropriate
antibiotic and incubate overnight at 37
C. If a control experiment lacking one of the fragments was included, significantly
more colonies should be obtained in the presence of all inserts
than in the negative control.
5. Purify plasmid DNA from 6 to 12 colonies for restriction
analysis and sequencing. We typically first digest 500 ng of
purified plasmid with BamHI/XbaI and XhoI/NheI (both in
CutSmart Buffer) to control the presence of the desired inserts
(see Note 6). In the second step, two positive clones are
selected, and their expression cassettes are sequenced (see
sequencing oligos in Table 4).
3.5 Gibson
Assembly
® ,
NEBuilder
® HiFi DNA
Assembly, or
In-Fusion
® HD Cloning
Since SLIC is a simple and cost-effective technology, it is usually
tested by default in our laboratory. However, the assembly of some
constructs may prove more challenging than others, and it does not
always yield successful constructs through SLIC. In these cases, we
try commercial technologies such as NEBuilder HiFi (NEB) or
Production of Multiprotein Complexes Using the Baculovirus Expression. . .
31
As mentioned above, different approaches can be used to assemble
DNA fragments into a linearized vector containing overlapping
ends. Sequence and ligation–independent cloning (SLIC), in contrast to most other homology-based cloning strategies, does not
necessitate specific kits and only requires T4 DNA polymerase to be
tested first.
1. Set up the T4 DNA polymerase treatment for the four PCR
products in separate tubes. In each tube, mix 1 μg of DNA,
3 μL of 10Â NEB 2.1 buffer, and 0.5 U of T4 DNA Polymerase in a final volume of 30 μL. Incubate the reaction for 30 min
at room temperature (see Note 4).
2. Stop the exonuclease reaction by adding 3 μL of a 10 mM
dCTP solution (1/10 of the reaction volume) and leave on
ice. In the absence of dNTPs, the T4 DNA polymerase has a 3
0
to 5
0 exonuclease activity and “chews” the DNA leaving a
single-stranded region. Addition of dCTP stops the exonuclease activity and shifts it to a polymerase activity at the first
occurrence of this nucleotide.
3. Prepare an annealing mix containing the polymerase-treated
vector backbone, the dual promoter unit, and the two cDNAs
in a 1:2:2:2 ratio using 250 ng (0.15 pmoles) of a 3 kb vector
and appropriate amount of inserts (see Note 5), in a total
volume of 20 μL. Incubate the annealing mix at 37
C for
30 min and leave on ice until transformation or store at
À20
C. A negative control where one of the insert is omitted
can be included to evaluate background.
4. Use 10 μL of the annealing reaction to transform 100 μL of
E. coli competent cells (do not forget to use Pir1 cells for
plasmids with an R6Kγ replication origin). Plate the transformation mixture on LB agar plates containing the appropriate
antibiotic and incubate overnight at 37
C. If a control experiment lacking one of the fragments was included, significantly
more colonies should be obtained in the presence of all inserts
than in the negative control.
5. Purify plasmid DNA from 6 to 12 colonies for restriction
analysis and sequencing. We typically first digest 500 ng of
purified plasmid with BamHI/XbaI and XhoI/NheI (both in
CutSmart Buffer) to control the presence of the desired inserts
(see Note 6). In the second step, two positive clones are
selected, and their expression cassettes are sequenced (see
sequencing oligos in Table 4).
3.5 Gibson
Assembly
® ,
NEBuilder
® HiFi DNA
Assembly, or
In-Fusion
® HD Cloning
Since SLIC is a simple and cost-effective technology, it is usually
tested by default in our laboratory. However, the assembly of some
constructs may prove more challenging than others, and it does not
always yield successful constructs through SLIC. In these cases, we
try commercial technologies such as NEBuilder HiFi (NEB) or
Production of Multiprotein Complexes Using the Baculovirus Expression. . .
31