StyI-HF®

Protocol tips

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	We performed both single and double digestion. For single digestion we used the 4-base cutter MseI (NEB, R0525S.) For double digestion we used MseI and the 6-base cutter, StyI-HF (NEB, R3500S.) The digestion reaction steps are as following: Normalize the cleaned double stranded cDNA to the appropriate concentration (~30ng/μl.) Dilute MseI (10U/μl) and Styl-HF (20U/μl) enzyme in dilutant A solution (NEB B8001S) to the ratio 1:8 and 1:14, respectively. Set up a 30μl restriction digestion reaction as following: 10μl double-stranded cDNA (a total of 300ng), 3μl Cutsmart buffer (NEB), 1μl diluted MseI, 1μl diluted Styl-HF (substitute with H20 in case of single digestion), and 15μl H2O. In a Thermocycler, incubate the digestion reaction at 37°C for 4 hours, 65°C for 20 min, and hold at 4°C. Proceed immediately to the next step. The sized distribution pattern of ds-cDNA, MseI digested cDNA, Styl digested cDNA, and MseI-Styl double digested cDNA are shown in Fig 4A, 4B, 4C and 4D, respectively.

Protocol tips

We performed both single and double digestion. For single digestion we used the 4-base cutter MseI (NEB, R0525S.) For double digestion we used MseI and the 6-base cutter, StyI-HF (NEB, R3500S.) The digestion reaction steps are as following: Normalize the cleaned double stranded cDNA to the appropriate concentration (~30ng/μl.) Dilute MseI (10U/μl) and Styl-HF (20U/μl) enzyme in dilutant A solution (NEB B8001S) to the ratio 1:8 and 1:14, respectively. Set up a 30μl restriction digestion reaction as following: 10μl double-stranded cDNA (a total of 300ng), 3μl Cutsmart buffer (NEB), 1μl diluted MseI, 1μl diluted Styl-HF (substitute with H20 in case of single digestion), and 15μl H2O. In a Thermocycler, incubate the digestion reaction at 37°C for 4 hours, 65°C for 20 min, and hold at 4°C. Proceed immediately to the next step. The sized distribution pattern of ds-cDNA, MseI digested cDNA, Styl digested cDNA, and MseI-Styl double digested cDNA are shown in Fig 4A, 4B, 4C and 4D, respectively.

Publication protocol

"We performed both single and double digestion. For single digestion we used the 4-base cutter MseI (NEB, R0525S.) For double digestion we used MseI and the 6-base cutter, StyI-HF (NEB, R3500S.) The digestion reaction steps are as following:

Normalize the cleaned double stranded cDNA to the appropriate concentration (~30ng/μl.) Dilute MseI (10U/μl) and Styl-HF (20U/μl) enzyme in dilutant A solution (NEB B8001S) to the ratio 1:8 and 1:14, respectively. Set up a 30μl restriction digestion reaction as following:

10μl double-stranded cDNA (a total of 300ng), 3μl Cutsmart buffer (NEB), 1μl diluted MseI, 1μl diluted Styl-HF (substitute with H20 in case of single digestion), and 15μl H2O. In a Thermocycler, incubate the digestion reaction at 37°C for 4 hours, 65°C for 20 min, and hold at 4°C. Proceed immediately to the next step. The sized distribution pattern of ds-cDNA, MseI digested cDNA, Styl digested cDNA, and MseI-Styl double digested cDNA are shown in Fig 4A, 4B, 4C and 4D, respectively"

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