MseI NEB#R0525

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	genomic DNA from each sample was treated with NdeI, MseI (NEB, Ipswich, MA, USA), T4 DNA ligase (NEB), ATP (NEB), and MseI adapter at 37 °C. These restriction-ligation reaction solutions were diluted and mixed with dNTP, Taq DNA polymerase (NEB) and MseI primer containing barcode 1 for PCR reactions. The E.Z.N.A. Cycle Pure Kit (Omega, London, UK) were used to purify the PCR products. The purified PCR products were pooled and incubated at 37 °C with MseI, T4 DNA ligase, ATP, and Solexa adapter.	After incubation, the reaction products were then purified using a Quick Spin column (Qiagen, Venlo, Netherlands), and electrophoresed on a 2% agarose gel.

Protocol tips
genomic DNA from each sample was treated with NdeI, MseI (NEB, Ipswich, MA, USA), T4 DNA ligase (NEB), ATP (NEB), and MseI adapter at 37 °C. These restriction-ligation reaction solutions were diluted and mixed with dNTP, Taq DNA polymerase (NEB) and MseI primer containing barcode 1 for PCR reactions. The E.Z.N.A. Cycle Pure Kit (Omega, London, UK) were used to purify the PCR products. The purified PCR products were pooled and incubated at 37 °C with MseI, T4 DNA ligase, ATP, and Solexa adapter.
Downstream tips
After incubation, the reaction products were then purified using a Quick Spin column (Qiagen, Venlo, Netherlands), and electrophoresed on a 2% agarose gel.

Publication protocol

SLAF-seq was used to genotype a total of 149 individuals, and the two parents, as previously described [13], with a few modifications. In brief, genomic DNA from each sample was treated with NdeI, MseI (NEB, Ipswich, MA, USA), T4 DNA ligase (NEB), ATP (NEB), and MseI adapter at 37 °C. These restriction-ligation reaction solutions were diluted and mixed with dNTP, Taq DNA polymerase (NEB) and MseI primer containing barcode 1 for PCR reactions. The E.Z.N.A. Cycle Pure Kit (Omega, London, UK) were used to purify the PCR products. The purified PCR products were pooled and incubated at 37 °C with MseI, T4 DNA ligase, ATP, and Solexa adapter. After incubation, the reaction products were then purified using a Quick Spin column (Qiagen, Venlo, Netherlands), and electrophoresed on a 2% agarose gel. SLAFs of 550–600 bp (including adapter sequence indexes and adaptors) in size were isolated using Gel Extraction Kits (Qiagen). These SLAFs were then subjected to PCR with Phusion Master Mix (NEB) and Solexa Amplification primer mix to add barcode 2. PCR products were gel purified and SLAFs of 330–380 bp selected for paired-end sequencing on an Illumina HiSeq 2500 sequencing platform (Illumina, San Diego, CA, USA). DNA sequence reads were 200 nucleotides long.

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