BbsI-HF®

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Approximately 3 μg PCR products in 250 μl of 1× CutSmart buffer was mixed with 50 U BbsI-HF, 20 U rSAP, and incubated at 37 °C for 2 h, followed by deactivation of rSAP at 80 °C for 20 min. MluCI (20 U) was subsequently added to the mixture and the digestion was continued at 37 °C for 1 h. The resulting mixture was extracted once with phenol/chloroform/isoamyl alcohol (25:24:1, v/v) and the aqueous phase was evaporated, desalted by Waters Oasis HLB extraction cartridges (Milford, MA), and redissolved in 10 μl water.

Protocol tips

Approximately 3 μg PCR products in 250 μl of 1× CutSmart buffer was mixed with 50 U BbsI-HF, 20 U rSAP, and incubated at 37 °C for 2 h, followed by deactivation of rSAP at 80 °C for 20 min. MluCI (20 U) was subsequently added to the mixture and the digestion was continued at 37 °C for 1 h. The resulting mixture was extracted once with phenol/chloroform/isoamyl alcohol (25:24:1, v/v) and the aqueous phase was evaporated, desalted by Waters Oasis HLB extraction cartridges (Milford, MA), and redissolved in 10 μl water.

Publication protocol

"Approximately 3 μg PCR products in 250 μl of
1× CutSmart buffer was mixed with 50 U BbsI-HF,
20 U rSAP, and incubated at 37 °C for 2 h, followed
by deactivation of rSAP at 80 °C for 20 min. MluCI
(20 U) was subsequently added to the mixture and
the digestion was continued at 37 °C for 1 h. The
resulting mixture was extracted once with
phenol/chloroform/isoamyl alcohol (25:24:1, v/v)
and the aqueous phase was evaporated, desalted by
Waters Oasis HLB extraction cartridges (Milford,
MA), and redissolved in 10 μl water. A 5-μl aliquot
was analyzed by LC-MS/MS on an LTQ linear ion
trap mass spectrometer (Thermo Electron, San Jose,
CA, USA) with an Agilent Zorbax SB-C18 column
(0.5 × 150 mm, 5 μm in particle size). The gradient
was 5 min of 5–20% methanol followed by 35 min
of 20–50% methanol in 400 mM HFIP (pH was
adjusted to 7.0 with triethylamine). The
temperature for the ion-transfer tube was 300 °C,
and the mass spectrometer was set up for acquiring
the higher-resolution ‘ultra-zoom scan’ MS and
full-scan MS/MS for the [M-3H]3- ions of 10 mer
ODNs, d(GGCMNGCTAT), with ‘M’ and ‘N’
being A, T, C and G."

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