Page 14 - Macromolecular Crystallography

CLASSICAL CLONING, EXPRESSION, AND PURIFICATION 3

Protocol 1.1 Construction of recombinant vector by PCR

1. Digest the vector with specific restriction enzymes to 5. Digest the insert with specific restriction enzymes.
generate ends compatible for ligation with the coding To a microcentrifuge tube add:
sequence to be cloned.
2. Purify using preparative agarose gel electrophoresis. 5 µl of appropriate 10× restriction enzyme buffer
3. Extract from agarose using a commercial gel 0.5 µl 100× BSA
extraction kit. 0.2 µg DNA
4. Amplify the insert sequence using suitable 2.5 µl of restriction enzyme(s)
oligonucleotide primers. Sterile water to a volume of 50 µl
To a PCR tube on ice, add:
Incubate the reaction mixture for 2–4 h at the temperature
5 µl10× PCR reaction buffer appropriate for the restriction enzyme used.
5 µl dNTP mix (2 mM each dATP, dCTP, dGTP, dTTP) Purify using either agarose or a commercial kit.
5 µl of each forward and reverse primers (10 pmol/µl) (If the two enzymes do not have a compatible buffer,
0.5 µl DNA template (100–250 ng for mammalian perform the digestion in two steps, purifying the insert after
genomic DNA, each step.)
20 ng for linearized plasmid DNA) 6. Ligate vector and gene product
To a microcentrifuge tube add:
1.0 µl Taq DNA polymerase
2–8 µl 25 mM MgCl 2 solution 100 ng of digested vector DNA
28.5 µl sterile water
Insert fragment (1:1 to 3:1 molar ratio of the insert to the
Set up negative control reactions omitting primers or DNA vector)
substrate. 4 µl5× ligation buffer
Amplify DNA for 25 cycles with the appropriate sequence 1 µl T4 DNA ligase
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of melting (95 C for 1 min), annealing temperature (to be
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calculated from the melting temperatures of the primers Incubate at 16 C for 4–16 h or at 4 C overnight.
used), and replication (72 C for 1 min), followed by a final 7. Transform competent E. coli host with recombinant
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10 min extension step at 72 C. vector and select for recombinants by antibiotic resistance
Purify amplified DNA using a commercial kit (Qiagen appropriate for the plasmid.
QiaQuick). 8. Identify colonies by PCR or plasmid mini-preps.
Determine the concentration of the insert. 9. DNA sequence the construct.

usually necessary unless PCR introduces contami- Thecellsarefirstmadecompetentfortransformation
nating sequences. by treatment with calcium chloride using a stan-
The PCR product is then digested with appropri- dard procedure (Appelbaum and Shatzman, 1999).
ate restriction enzymes, unless it is to be first ligated Recombinants containing the inserted gene can be
into a TA cloning vector. (TA cloning involves two conveniently screened by PCR, using vector-specific
stages – firstly cloning into a TA vector followed by and gene-specific primers.
subcloning into an expression vector.) It is important Directional cloning (by ligation into two dif-
to ensure that the insert is properly digested, and ferent restriction sites) is usually the preferred
when carrying out a simultaneous double digestion option, having the advantage of not requiring
the enzymes must be compatible with the buffer dephosphorylation of the vector and also avoiding
supplied. Before the ligation step the insert should possibility of the product ending up in the wrong
be purified, either by electrophoresis on agarose or orientation. Finally, it is important to sequence the
with a commercial kit. It is always good practice construct in order to identify any mutations that
to carry out a controlled ligation with the vector may have been generated during the PCR reaction.
alone. The recombinant ligated vector is next intro- Protocol 1.1 outlines the sequence of steps involved
duced into the selected host strain (Section 1.2.2.1). in the construction of a recombinant vector. Specific