PstI-HF®

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	High-fidelity (HF) ApoI and PstI restriction enzymes were obtained from New England BioLabs Inc. (Ipswich, Massachusetts USA). The optimization of restriction enzyme digestion (Supplementary Fig. 4) was performed on 500 ng of FLO1 cell line genomic DNA and included optimization of enzyme concentration, library purification procedure, PCR cycle optimization and removal of FFPE artefacts.

Protocol tips
High-fidelity (HF) ApoI and PstI restriction enzymes were obtained from New England BioLabs Inc. (Ipswich, Massachusetts USA). The optimization of restriction enzyme digestion (Supplementary Fig. 4) was performed on 500 ng of FLO1 cell line genomic DNA and included optimization of enzyme concentration, library purification procedure, PCR cycle optimization and removal of FFPE artefacts.

Publication protocol

"Both restriction digestion and ligation reaction were performed simultaneously. 500 ng of genomic DNA was digested with 50 U of PstI-HF and ApoI-HF in presence of 0.187 mM mutREAD i5 and i7 adapters, 400 U of T4 ligase and 1 mM ATP in 1X CutSmart buffer. The reaction was incubated on a thermal cycler at 30 °C for 3 h. Ligation reaction was stopped by addition of 10 µl of 50 mM EDTA.

Two step size selection for 400–500 bp inserts (DNA fragments, excluding adapters) was performed using Agencourt AMPure XP beads (BECKMAN COULTER, Brea California US). Unwanted larger fragments were removed with 0.6× ratio of AMPure beads to ligation product and the short fragments were removed by 0.15× size selection.

The size selected DNA fragments ligated with adapters (20 μl) were amplified using PCR primers (i5nn/i7nn) compatible with Illumina sequencing platform. The reaction was performed in total volume of 100 µl with 0.8 U of Phusion high-fidelity polymerase, in the presence of 0.2 mM dNTPs and 1X Phusion High Fidelity buffer. PCR was performed in the following conditions: 98 °C/2 min denaturation, 12 cycles of amplification at 98 °C/10 s, 65 °C/30 s, 72 °C/30 s and final extension at 72 °C for 5 min. Libraries were purified using 0.8X AMPure beads (80 μl beads + 100 μl library), this step was repeated one more time to remove all unwanted leftover reactants during PCR. Libraries were eluted in 20 μl TE buffer (Tris-EDTA buffer 10 mM Tris-HCl and 0.1 mM EDTA, pH 8) and stored at −20 °C. Quality control was performed on Agilent 2100 Bioanalyzer using Agilent High Sensitivity DNA kit (Santa Clara, California, US) or High Sensitivity D1000 TapeStation kit (Agilent). Quantification of the libraries was performed using KAPA Library Quantification kit (KK4953-07960573001 for Illumina platforms, Kapa Biosysytems Roche Holding AG Basel Switzerland) on the Light cycler 480 (Roche Life Sciences, Basel Switzerland). Libraries with unique adapters were pooled and sequenced on the HiSeq4000 using paired end, 150 bps chemistry."

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Restriction Enzymes PstI using PstI-HF® from New England BioLabs.

Manufacturer protocol

Download the product protocol from New England BioLabs for PstI-HF® below.

Videos

Check out videos that might be relevant for performing Restriction Enzymes PstI using PstI-HF® from New England BioLabs. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.