Bio-Rad Profinity eXact Purification Resin - Manual (Page 19)

Forward and reverse PCR primers starting with the predefined eight bases
illustrated in Step 1 are used to PCR the target gene. Using the 3'
Æ5'
exonuclease activity of T4 DNA polymerase, the PCR product is treated
with T4 DNA polymerase in the presence of dGTP to create overhangs
compatible with the RIC-ready pPAL7 vector. The prepared insert and
RIC-ready vector are then ligated using standard ligation techniques and
finally transformed into an appropriate
Escherichia coli cloning host such
as the E. coli C-Max5a competent cells provided with the Profinity eXact
system.
Method
1. PCR-amplify target gene using iProof ™ high-fidelity DNA
polymerase.
Both forward and reverse primers must be 5'-phosphorylated to
clone into the dephosphorylated vector, and they must contain the
following DNA sequences (
N indicates target DNA sequence):
Thr-Ser
Forward primer: 5' – P-AAGCTTTG(ACTTCT)NNNNNNNNN… – 3'
Optional
stop
Reverse primer: 5' – P-AATTCTTANNNNNNNNN… – 3'
The optional ACTTCT (Thr-Ser) spacer of the forward primer can be
included to minimize all target protein effects on cleavage of the
Profinity eXact tag. If the template is an ampicillin-resistant plasmid, it
may be DpnI-treated post PCR amplification to ensure background-free
transformants.
2. Purify, if desired, PCR product by either excising and purifying
band following agarose gel electrophoresis, or using PCR
Kleen
™ spin column kit (catalog #732-6000).
3. Generate RIC-ready pPAL7 vector-compatible overhangs by T4
DNA polymerase treatment of purified PCR product.
16
10011260A.qxp 11/12/2007 11:31 AM Page 16