sybrgreen i  (New England Biolabs)


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    Structured Review

    New England Biolabs sybrgreen i
    Sybrgreen I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 82/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sybrgreen i/product/New England Biolabs
    Average 82 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sybrgreen i - by Bioz Stars, 2019-10
    82/100 stars

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    Amplification:

    Article Title: Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming
    Article Snippet: Purified PCR products with the four probe sets on the same template DNA were pooled in equal molar ratio, and reamplified in 4× 100 μl reactions with 4-μl template (10~15 ng/μl), 200 μM dNTPs, 20 μM dUTP, 200 nM AmpF6.3 primer, 200 nM AmpR6.3 primer, 0.4× SybrGreen I and 200 μl 2× Taq Master Mix (NEB) at 94 °C for 3 min, 8 cycles of 94 °C for 45 s, 55 °C for 45 s, 72 °C for 45 s and 72 °C for 3 min. PCR amplicons were purified with Qiaquick columns, and digested with Mme I: ~3.6 nmole purified PCR amplicons, 16 units of Mme I (2 U/μl; NEB), 100 μM SAM in 1× NEB Buffer 4 at 37 °C for 1 h. The digestions were again column purified, and digested with 3 U USER enzyme (1 U/μl) at 37 °C for 2 h, then with 10 units S1 nuclease (10 U/μl; Invitrogen) in 1× S1 nuclease buffer at 37 °C for 10 min. .. Approximately 100–500 ng of the end-repaired DNA was ligated with 60 μM Solexa sequencing adaptors in 30 μl of 1× QuickLigase Buffer (NEB) with 1 μl QuickLigase for 15 min at 25 °C.

    Ligation:

    Article Title: Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming
    Article Snippet: Purified PCR products with the four probe sets on the same template DNA were pooled in equal molar ratio, and reamplified in 4× 100 μl reactions with 4-μl template (10~15 ng/μl), 200 μM dNTPs, 20 μM dUTP, 200 nM AmpF6.3 primer, 200 nM AmpR6.3 primer, 0.4× SybrGreen I and 200 μl 2× Taq Master Mix (NEB) at 94 °C for 3 min, 8 cycles of 94 °C for 45 s, 55 °C for 45 s, 72 °C for 45 s and 72 °C for 3 min. PCR amplicons were purified with Qiaquick columns, and digested with Mme I: ~3.6 nmole purified PCR amplicons, 16 units of Mme I (2 U/μl; NEB), 100 μM SAM in 1× NEB Buffer 4 at 37 °C for 1 h. The digestions were again column purified, and digested with 3 U USER enzyme (1 U/μl) at 37 °C for 2 h, then with 10 units S1 nuclease (10 U/μl; Invitrogen) in 1× S1 nuclease buffer at 37 °C for 10 min. .. Approximately 100–500 ng of the end-repaired DNA was ligated with 60 μM Solexa sequencing adaptors in 30 μl of 1× QuickLigase Buffer (NEB) with 1 μl QuickLigase for 15 min at 25 °C.

    Purification:

    Article Title: Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming
    Article Snippet: The amplicons of the expected size range (344–394 bp) were purified with 6% PAGE (6% TBE gel; Invitrogen). .. Purified PCR products with the four probe sets on the same template DNA were pooled in equal molar ratio, and reamplified in 4× 100 μl reactions with 4-μl template (10~15 ng/μl), 200 μM dNTPs, 20 μM dUTP, 200 nM AmpF6.3 primer, 200 nM AmpR6.3 primer, 0.4× SybrGreen I and 200 μl 2× Taq Master Mix (NEB) at 94 °C for 3 min, 8 cycles of 94 °C for 45 s, 55 °C for 45 s, 72 °C for 45 s and 72 °C for 3 min. PCR amplicons were purified with Qiaquick columns, and digested with Mme I: ~3.6 nmole purified PCR amplicons, 16 units of Mme I (2 U/μl; NEB), 100 μM SAM in 1× NEB Buffer 4 at 37 °C for 1 h. The digestions were again column purified, and digested with 3 U USER enzyme (1 U/μl) at 37 °C for 2 h, then with 10 units S1 nuclease (10 U/μl; Invitrogen) in 1× S1 nuclease buffer at 37 °C for 10 min. .. The fragmented DNA was column purified, and end repaired at 25 °C for 45 min in 25-μl reactions containing 2.5 μl 10× buffer, 2.5 μl dNTP mix (2.5 mMeach), 2.5 μl ATP (10 mM), 1 μl end-repair enzyme mix (Epicentre), and 15 μl DNA.

    Polymerase Chain Reaction:

    Article Title: Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming
    Article Snippet: The amplicons of the expected size range (344–394 bp) were purified with 6% PAGE (6% TBE gel; Invitrogen). .. Purified PCR products with the four probe sets on the same template DNA were pooled in equal molar ratio, and reamplified in 4× 100 μl reactions with 4-μl template (10~15 ng/μl), 200 μM dNTPs, 20 μM dUTP, 200 nM AmpF6.3 primer, 200 nM AmpR6.3 primer, 0.4× SybrGreen I and 200 μl 2× Taq Master Mix (NEB) at 94 °C for 3 min, 8 cycles of 94 °C for 45 s, 55 °C for 45 s, 72 °C for 45 s and 72 °C for 3 min. PCR amplicons were purified with Qiaquick columns, and digested with Mme I: ~3.6 nmole purified PCR amplicons, 16 units of Mme I (2 U/μl; NEB), 100 μM SAM in 1× NEB Buffer 4 at 37 °C for 1 h. The digestions were again column purified, and digested with 3 U USER enzyme (1 U/μl) at 37 °C for 2 h, then with 10 units S1 nuclease (10 U/μl; Invitrogen) in 1× S1 nuclease buffer at 37 °C for 10 min. .. The fragmented DNA was column purified, and end repaired at 25 °C for 45 min in 25-μl reactions containing 2.5 μl 10× buffer, 2.5 μl dNTP mix (2.5 mMeach), 2.5 μl ATP (10 mM), 1 μl end-repair enzyme mix (Epicentre), and 15 μl DNA.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming
    Article Snippet: Purified PCR products with the four probe sets on the same template DNA were pooled in equal molar ratio, and reamplified in 4× 100 μl reactions with 4-μl template (10~15 ng/μl), 200 μM dNTPs, 20 μM dUTP, 200 nM AmpF6.3 primer, 200 nM AmpR6.3 primer, 0.4× SybrGreen I and 200 μl 2× Taq Master Mix (NEB) at 94 °C for 3 min, 8 cycles of 94 °C for 45 s, 55 °C for 45 s, 72 °C for 45 s and 72 °C for 3 min. PCR amplicons were purified with Qiaquick columns, and digested with Mme I: ~3.6 nmole purified PCR amplicons, 16 units of Mme I (2 U/μl; NEB), 100 μM SAM in 1× NEB Buffer 4 at 37 °C for 1 h. The digestions were again column purified, and digested with 3 U USER enzyme (1 U/μl) at 37 °C for 2 h, then with 10 units S1 nuclease (10 U/μl; Invitrogen) in 1× S1 nuclease buffer at 37 °C for 10 min. .. Approximately 100–500 ng of the end-repaired DNA was ligated with 60 μM Solexa sequencing adaptors in 30 μl of 1× QuickLigase Buffer (NEB) with 1 μl QuickLigase for 15 min at 25 °C.

    Sequencing:

    Article Title: Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming
    Article Snippet: Purified PCR products with the four probe sets on the same template DNA were pooled in equal molar ratio, and reamplified in 4× 100 μl reactions with 4-μl template (10~15 ng/μl), 200 μM dNTPs, 20 μM dUTP, 200 nM AmpF6.3 primer, 200 nM AmpR6.3 primer, 0.4× SybrGreen I and 200 μl 2× Taq Master Mix (NEB) at 94 °C for 3 min, 8 cycles of 94 °C for 45 s, 55 °C for 45 s, 72 °C for 45 s and 72 °C for 3 min. PCR amplicons were purified with Qiaquick columns, and digested with Mme I: ~3.6 nmole purified PCR amplicons, 16 units of Mme I (2 U/μl; NEB), 100 μM SAM in 1× NEB Buffer 4 at 37 °C for 1 h. The digestions were again column purified, and digested with 3 U USER enzyme (1 U/μl) at 37 °C for 2 h, then with 10 units S1 nuclease (10 U/μl; Invitrogen) in 1× S1 nuclease buffer at 37 °C for 10 min. .. The fragmented DNA was column purified, and end repaired at 25 °C for 45 min in 25-μl reactions containing 2.5 μl 10× buffer, 2.5 μl dNTP mix (2.5 mMeach), 2.5 μl ATP (10 mM), 1 μl end-repair enzyme mix (Epicentre), and 15 μl DNA.

    Shotgun Sequencing:

    Article Title: Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming
    Article Snippet: Paragraph title: Shotgun sequencing library construction ... Purified PCR products with the four probe sets on the same template DNA were pooled in equal molar ratio, and reamplified in 4× 100 μl reactions with 4-μl template (10~15 ng/μl), 200 μM dNTPs, 20 μM dUTP, 200 nM AmpF6.3 primer, 200 nM AmpR6.3 primer, 0.4× SybrGreen I and 200 μl 2× Taq Master Mix (NEB) at 94 °C for 3 min, 8 cycles of 94 °C for 45 s, 55 °C for 45 s, 72 °C for 45 s and 72 °C for 3 min. PCR amplicons were purified with Qiaquick columns, and digested with Mme I: ~3.6 nmole purified PCR amplicons, 16 units of Mme I (2 U/μl; NEB), 100 μM SAM in 1× NEB Buffer 4 at 37 °C for 1 h. The digestions were again column purified, and digested with 3 U USER enzyme (1 U/μl) at 37 °C for 2 h, then with 10 units S1 nuclease (10 U/μl; Invitrogen) in 1× S1 nuclease buffer at 37 °C for 10 min.

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  • News
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  • Team
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  • 82
    New England Biolabs sybrgreen i
    Sybrgreen I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 82/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sybrgreen i/product/New England Biolabs
    Average 82 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sybrgreen i - by Bioz Stars, 2019-10
    82/100 stars
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