Biomedical Engineering Reference
In-Depth Information
3.5 Destination
Vector Construction
1. Cassettes for generating new Destination vectors generally
contain two
attR
sites fl anking a set of positive and negative
selection markers. In the case of standard Gateway vectors,
the negative selection is done by the ccdB gene, and the posi-
tive selection by the CAT gene for chloramphenicol resistance.
2. To generate new Destination vectors, transfer vectors contain-
ing attR4-attR2 (pSpcRFA42) or attR4-attR3 (pSpcRFA43)
cassettes fl anked by a pair of EcoRV restriction sites can be
used. Digest with EcoRV produces a Gateway cassette which
can be introduced into any vector with a blunt cutting site, or
which has been blunt-ended.
3. Prepare Destination vector cassette by digesting a suitable
amount of the parental vector with EcoRV, and purifi cation
using the QIAquick PCR Purifi cation Kit. This cut material
can be saved for future use, so we suggest doing a single large
reaction and storing the cut material at −20 °C for subsequent
vector conversion work (
see
Note 20
).
4. For production of Destination vectors using a blunt cutting
site, 1
g of the vector of interest must fi rst be linearized using
a restriction endonuclease that generates blunt ends. After
digestion, 2
μ
L of the linearized product should be confi rmed
on a 0.8 % agarose gel to verify size by comparison to a linear
DNA standard such as the ReadyLoad 1 kb DNA ladder (
see
Note 21).
Purify the linearized vector using the QIAquick
PCR Purifi cation Kit following the manufacturer's protocol
and elute the DNA in 50
μ
L.
5. For production of Destination vectors using a cohesive end cut
site, you will require enzymatic blunting to allow correct inser-
tion of the Gateway cassette fragment. The purifi ed linearized
vector with cohesive ends must be treated with DNA
Polymerase 1, Klenow fragment (for 5
μ
′
DNA overhangs), or
T4 DNA Polymerase (for 3
DNA overhangs). Standard man-
ufacturer's protocols (New England Biolabs, Beverly, MA) can
be used for both reactions. QiaQuick PCR Purifi cation and
subsequent 50
′
L elution is used following the Klenow proto-
col, whereas the product of the NEB Quick Blunting Reaction
is used directly for Step 5.
6. Use 1
μ
L of the blunt-ended linearized vector in a ligation
reaction with 3
μ
L of the digested Gateway cassette of choice.
Bring the reaction to 10
μ
μ
L with water, and add 10
μ
L of the
L of T4 DNA
Ligase. Incubate the reaction at 25 °C for 10 min (
see
Note 22
).
Based on concentrations of the vector and insert, these amounts
may vary, with the goal of a 1:3 vector:insert ratio.
7. Add 1
10× Ligase Reaction Buffer, followed by 1
μ
L of the ligation reaction to a microcentrifuge tube
containing 20
μ
L of chemically competent
E. coli ccdB
Survival
cells and incubate on ice for 10-20 min. (
see
Note 8
).
μ
